Methods and compositions for amplification of RNA sequences

ABSTRACT

The invention provides methods for linear and exponential amplification of RNA. They are particularly suitable for amplifying a plurality of RNA species in a sample. The methods are based on hybridization of polynucleotide comprising a propromoter sequence to a primer extension product to generate an intermediate polynucleotide capable of driving transcription, whereby multiple copies of RNA products comprising sequences complementary to an RNA sequence of interest are generated. The methods are useful for preparation of nucleic acid libraries and substrates for analysis of gene expression of cells in biological samples. The invention also provides compositions and kits for practicing the amplification methods, as well as methods which use the amplification products.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of the provisionalpatent application U.S. Ser. No. 60/274,236, filed Mar. 9, 2001, whichis incorporated by reference in its entirety.

TECHNICAL FIELD

[0002] The invention relates to the field of polynucleotideamplification. More particularly, the invention provides methods,compositions and kits for amplifying (i.e., making multiple copies) RNAsequences of interest which employ a polynucleotide comprising apropromoter and RNA transcription.

BACKGROUND ART

[0003] The ability to amplify ribonucleic acid (RNA) is an importantaspect of efforts to elucidate biological processes. To date, RNA(generally, mRNA) amplification is most commonly performed using thereverse transcriptase-polymerase chain reaction (RT-PCR) method andvariations thereof. These methods are based on replication of RNA byreverse transcriptase to form single stranded DNA complementary to theRNA (cDNA), which is followed by polymerase chain reaction (PCR)amplification to produce multiple copies of double stranded DNA.Although these methods are most commonly used, they have somesignificant drawbacks: a) the reactions require thermocycling; b) theproducts are double stranded, thus rendering them less accessible tobinding to probes; c) the reactions are prone to contamination withproducts of prior amplification, thus requiring strict containment ofreaction mixtures; and d) the exponential nature of amplification ofthese methods renders them prone to generate pools of products which donot truly reflect the representation of the various RNA sequences in theinput total RNA sample, due to unequal efficiency of amplification ofdifferent sequences, and the nature of exponential amplification whichis based on replication of amplification products rather than oncontinued replication of the input target RNAs.

[0004] The total cellular mRNA represents gene expression activity at adefined time. Gene expression is affected by cell cycle progression,developmental regulation, response to internal and external stimuli andthe like. The profile of expressed genes for any cell type in anorganism reflects normal or disease states, response to various stimuli,developmental stages, cell differentiation, and the like.

[0005] Various methods for the analysis of gene expression have bedeveloped in recent years. See, for example, U.S. Pat. Nos. 5,744,308;6,143,495; 5,824,517; 5,829,547; 5,888,779; 5,545,522; 5,716,785;5,409,818; EP 0971039A 2; EP 0878553A 2. These include quantification ofspecific mRNAs, and the simultaneous quantification of a large number ofmRNAs, as well as the detection and quantification of patterns ofexpression of known and unknown genes. The analysis of gene expressionprofiles is currently one of the most powerful tools in the study ofcellular differentiation and cellular development, and in theinvestigation of normal and disease states of various organisms, inparticular in human. This analysis is crucial for gene discovery,molecular medicine and drug discovery processes.

[0006] Essential for gene expression profiling is the ability torandomly amplify the total cellular mRNAs prepared from any cell ortissue. Although analysis of non-amplified mRNA is feasible, asignificant amount of starting mRNA would be required. However, thetotal amount of sample mRNA that is available is frequently limited bythe amount of biological sample from which it is derived. Biologicalsamples are often limited in amount and precious. Moreover, the amountof the various mRNA species is not equal; some species are more abundantthan others, and these are more likely and easier, to analyze. Theability to amplify mRNA sequences enables the analysis of less abundant,rare mRNA species. The ability to analyze small samples, by means ofnucleic acid amplification, is also advantageous for design parametersof large scale screening of effector molecule libraries, for whichreduction in sample volume is a major concern both for the ability toperform very large scale screening or ultra high throughput screening,and in view of the limiting amounts of library components.

[0007] Therefore, there is a need for improved RNA amplification methodsthat overcome drawbacks in existing methods. The invention providedherein fulfills this need and provides additional benefits.

[0008] All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

DISCLOSURE OF THE INVENTION

[0009] The invention provides methods, compositions, and kits forpolynucleotide, specifically ribonucleic acid, amplification, as well asapplications of the amplification methods.

[0010] In one aspect, the invention provides methods of generatingmultiple copies of the complementary sequence of an RNA sequence ofinterest, said method comprising the steps of: (a) extending a firstprimer hybridized to a target RNA with an RNA-dependent DNA polymerase,whereby a complex comprising a first primer extension product and thetarget RNA is produced; (b) cleaving RNA in the complex of step (b) withan enzyme that cleaves RNA from an RNA/DNA hybrid; (c) extending asecond primer hybridized to the first primer extension product with aDNA-dependent DNA polymerase, whereby a complex comprising the firstprimer extension product and a second primer extension product isproduced; (d) denaturing the complex of step (c); and (e) hybridizing tothe second primer extension product a propromoter polynucleotidecomprising a propromoter and a region which hybridizes to the secondprimer extension product under conditions which allow transcription tooccur by RNA polymerase, such that RNA transcripts are producedcomprising sequences complementary to the target RNA; whereby multiplecopies of the complementary sequence of the RNA sequence of interest aregenerated.

[0011] In one aspect, the invention provides methods of generatingmultiple copies of (amplifying) the complementary sequence of an RNAsequence of interest, said method comprising the steps of: (a)hybridizing a first primer to a target ribonucleic acid; (b) extendingthe first primer with an RNA-dependent DNA polymerase, whereby a complexcomprising a first primer extension product and the target ribonucleicacid is produced; (c) cleaving ribonucleic acid in the complex of step(b) with an enzyme that cleaves RNA from an RNA/DNA hybrid; (d)hybridizing a second primer to the first primer extension product; (e)extending the second primer with a DNA-dependent DNA polymerase, wherebya complex comprising the first primer extension product and a secondprimer extension product is produced; (f) denaturing the complex of step(e); (g) hybridizing to the second primer extension product apolynucleotide comprising a propromoter and a region which hybridizes tothe second primer extension product under conditions which allowtranscription to occur by RNA polymerase, such that RNA transcripts areproduced comprising sequences complementary to the target ribonucleicacid, whereby multiple copies of the complementary sequence of the RNAsequence of interest are generated. In some embodiments, thepolynucleotide comprising a propromoter is a propromoter templateoligonucleotide (PTO). In some embodiments, the invention providesmethods of generating multiple copies of the complementary sequence ofan RNA sequence of interest, said methods comprising the steps of: (a)combining: a single stranded second primer extension product resultingfrom step (f) of the aspect of the invention described above; apropromoter polynucleotide comprising a propromoter and a region whichis hybridizable to a single stranded second primer extension product;and an RNA polymerase; and (b) incubating the mixture of step (a) underconditions (which includes necessary substrates and buffer conditions)that permit propromoter polynucleotide hybridization and RNAtranscription, whereby multiple copies of the complementary sequence ofthe RNA sequence of interest are generated. It is understood that anycombination of these incubation steps, and any single incubation step,to the extent that the incubation is performed as part of any of themethods described herein, fall within the scope of the invention. It isalso understood that methods that comprise one or more incubation stepsdo not require a separate combination step, as such combinations areimplicit in incubating the reaction mixture(s).

[0012] In another aspect, the invention provides methods of generatingmultiple copies of the complementary sequence of an RNA sequence ofinterest, said method comprising the steps of: (a) extending a firstprimer hybridized to a target RNA with an RNA-dependent DNA polymerase,whereby a complex comprising a first primer extension product and thetarget RNA is produced; (b) cleaving RNA in the complex of step (a) withan enzyme that cleaves RNA from an RNA/DNA hybrid; (c) extending asecond primer hybridized to the first primer extension product with aDNA-dependent DNA polymerase, whereby a complex comprising the firstprimer extension product and a second primer extension product isproduced; (d) denaturing the complex of step (c); (e) hybridizing to thesecond primer extension product a propromoter polynucleotide comprisinga propromoter and a region which hybridizes to the second primerextension product under conditions which allow transcription to occur byRNA polymerase, such that RNA transcripts are produced comprisingsequences complementary to the target RNA; (f) extending a third primerhybridized to said RNA transcripts with an RNA-dependent DNA polymerase,whereby a complex comprising a third primer extension product and an RNAtranscript is produced; (g) cleaving RNA in the complex of step (f) withan enzyme that cleaves RNA from an RNA/DNA hybrid; (h) hybridizing apropromoter polynucleotide comprising a propromoter and a region whichhybridizes to a single stranded third primer extension product underconditions which allow transcription to occur by RNA polymerase, suchthat RNA transcripts are produced comprising sequences complementary tothe target RNA; (i) optionally repeating steps (f) to (h); wherebymultiple copies of the complementary sequence of the RNA sequence ofinterest are produced.

[0013] In another aspect, the invention provides methods of generatingmultiple copies of (amplifying) the complementary sequence of an RNAsequence of interest, said method comprising the steps of: (a)hybridizing a first primer to a target ribonucleic acid; (b) extendingthe first primer with an RNA-dependent DNA polymerase, whereby a complexcomprising a first primer extension product and the target ribonucleicacid is produced; (c) cleaving ribonucleic acid in the complex of step(b) with an enzyme that cleaves RNA from an RNA/DNA hybrid; (d)hybridizing a second primer to the first primer extension product; (e)extending the second primer with a DNA-dependent DNA polymerase, wherebya complex comprising the first primer extension product and a secondprimer extension product is produced; (f) denaturing the complex of step(e); (g) hybridizing to the second primer extension product apropromoter and a region which hybridizes to the second primer extensionproduct under conditions which allow transcription to occur by RNApolymerase, such that RNA transcripts are produced comprising sequencescomplementary to the target ribonucleic acid; (h) hybridizing a thirdprimer to said RNA transcripts; (i) extending the third primer with anRNA-dependent DNA polymerase, whereby a complex comprising a thirdprimer extension product and an RNA transcript is produced; (j) cleavingRNA in the complex of step (i) with an enzyme that cleaves RNA from anRNA/DNA hybrid; (k) hybridizing a propromoter polynucleotide comprisinga propromoter and a region which hybridizes to a single stranded thirdprimer extension product under conditions which allow transcription tooccur by RNA polymerase, such that RNA transcripts are producedcomprising sequences complementary to the target ribonucleic acid; (l)optionally repeating steps (h) to (k), whereby multiple copies of thecomplementary sequence of the RNA sequence of interest are produced. Insome embodiments, the polynucleotide comprising a propromoter is apropromoter template oligonucleotide (PTO). In some embodiments, theinvention provides methods of generating multiple copies of thecomplementary sequence of an RNA sequence of interest, said methodscomprising the steps of: (a) combining: a single stranded second primerextension product resulting from step (f) described above in thisparagraph; a third primer comprising a sequence hybridizable to an RNAtranscript comprising a sequence complementary to the target RNA; apropromoter polynucleotide comprising a propromoter and a region whichis hybridizable to a single stranded second primer extension product; apropromoter polynucleotide comprising a propromoter and a region whichis hybridizable to a single stranded third primer extension product; anenzyme that cleaves RNA from an RNA/DNA hybrid; and an RNA polymerase;and (b) incubating the mixture of step (a) under conditions (whichincludes necessary substrates and buffers) that permit primer extension,RNA cleavage, propromoter polynucleotide hybridization and RNAtranscription, whereby multiple copies of the complementary sequence ofthe RNA sequence of interest are generated. In yet another embodiment,the invention provides methods of generating multiple copies of thecomplementary sequence of an RNA sequence of interest, said methodscomprising the steps of: (a) combining: an RNA transcript from step (g)described above in this paragraph; a third primer comprising a sequencehybridizable to the RNA transcript; a propromoter polynucleotidecomprising a propromoter and a region which is hybridizable to a singlestranded third primer extension product; an enzyme that cleaves RNA froman RNA/DNA hybrid; and an RNA polymerase; and (b) incubating the mixtureof step (a) under conditions (which includes necessary substrates andbuffers) that permit primer extension, RNA cleavage, propromoterpolynucleotide hybridization and RNA transcription, whereby multiplecopies of the complementary sequence of the RNA sequence of interest aregenerated.

[0014] In still another aspect, the invention provides methods ofgenerating multiple copies of (amplifying) the complementary sequence ofan RNA sequence of interest, said method comprising the steps of: (a)combining: a target ribonucleic acid; a first primer comprising asequence that is hybridizable to the target ribonucleic acid; a secondprimer comprising a sequence hybridizable to an extension product of thefirst primer; a propromoter polynucleotide comprising a propromoter anda region which is hybridizable to a single stranded second primerextension product; an RNA-dependent DNA polymerase; a DNA-dependent DNApolymerase; an RNA polymerase; and an enzyme that cleaves RNA from anRNA/DNA hybrid; and (b) incubating the mixture of step (a) underconditions (which includes necessary substrates and buffer conditions)that permit primer hybridization, primer extension, RNA cleavage,propromoter polynucleotide hybridization, and RNA transcription. In someembodiments, the polynucleotide comprising a propromoter is apropromoter template oligonucleotide (PTO).

[0015] In yet another aspect, the invention provides methods ofgenerating multiple copies of (amplifying) the complementary sequence ofan RNA sequence of interest, said method comprising the steps of: (a)combining: a target ribonucleic acid; a first primer comprising asequence that is hybridizable to the target ribonucleic acid; a secondprimer comprising a sequence hybridizable to an extension product of thefirst primer; a third primer comprising a sequence hybridizable to anRNA transcript comprising a sequence complementary to the targetribonucleic acid; a propromoter polynucleotide comprising a propromoterand a region which is hybridizable to a single stranded second primerextension product; a propromoter polynucleotide comprising a propromoterand a region which is hybridizable to a single stranded third primerextension product; an RNA-dependent DNA polymerase; a DNA-dependent DNApolymerase; an RNA polymerase; and an enzyme that cleaves RNA from anRNA/DNA hybrid; and (b) incubating the mixture of step (a) underconditions (which includes necessary substrates and buffer conditions)that permit primer hybridization, primer extension, RNA cleavage,propromoter polynucleotide hybridization, and RNA transcription. In someembodiments, the polynucleotide comprising a propromoter is apropromoter template oligonucleotide (PTO).

[0016] In another aspect, the invention provides methods of generatingmultiple copies of the complementary sequence of an RNA sequence ofinterest, said method comprising the steps of: (a) extending a firstprimer hybridized to a target RNA with an RNA-dependent DNA polymerase,whereby a complex comprising a first primer extension product and thetarget RNA is produced; (b) cleaving RNA in the complex of step (b) withan enzyme that cleaves RNA from an RNA/DNA hybrid; (c) extending asecond primer hybridized to the first primer extension product with aDNA-dependent DNA polymerase, whereby a complex comprising the firstprimer extension product and a second primer extension product isproduced; (d) denaturing the complex of step (c); and (e) hybridizing tothe second primer extension product a propromoter polynucleotidecomprising a propromoter and a region which hybridizes to the secondprimer extension product under conditions which allow transcription tooccur by RNA polymerase, such that RNA transcripts are producedcomprising sequences complementary to the target RNA; whereby multiplecopies of the complementary sequence of the RNA sequence of interest aregenerated.

[0017] In another aspect, the invention provides methods of generatingmultiple copies of the complementary sequence of an RNA sequence ofinterest, said method comprising the steps of: (a) extending a firstprimer hybridized to a target RNA with an RNA-dependent DNA polymerase,whereby a complex comprising a first primer extension product and thetarget RNA is produced; (b) cleaving RNA in the complex of step (a) withan enzyme that cleaves RNA from an RNA/DNA hybrid; (c) extending asecond primer hybridized to the first primer extension product with aDNA-dependent DNA polymerase, whereby a complex comprising the firstprimer extension product and a second primer extension product isproduced; (d) denaturing the complex of step (c); (e) hybridizing to thesecond primer extension product a propromoter polynucleotide comprisinga propromoter and a region which hybridizes to the second primerextension product under conditions which allow transcription to occur byRNA polymerase, such that RNA transcripts are produced comprisingsequences complementary to the target RNA; (f) extending a third primerhybridized to said RNA transcripts with an RNA-dependent DNA polymerase,whereby a complex comprising a third primer extension product and an RNAtranscript is produced; (g) cleaving RNA in the complex of step (f) withan enzyme that cleaves RNA from an RNA/DNA hybrid; (h) hybridizing apropromoter polynucleotide comprising a propromoter and a region whichhybridizes to a single stranded third primer extension product underconditions which allow transcription to occur by RNA polymerase, suchthat RNA transcripts are produced comprising sequences complementary tothe target RNA; (i) optionally repeating steps (f) to (h); wherebymultiple copies of the complementary sequence of the RNA sequence ofinterest are produced.

[0018] In another aspect, the invention provides a method of generatingmultiple copies of the complementary sequence of an RNA sequence ofinterest comprising incubating a reaction mixture, said reaction mixturecomprising: (a) a single stranded second primer extension productresulting from step (d) above; (b) a propromoter polynucleotidecomprising a propromoter and a region which is hybridizable to a singlestranded second primer extension product; and an RNA polymerase; whereinthe incubation is under conditions that permit propromoterpolynucleotide hybridization and RNA transcription, whereby multiplecopies of the complementary sequence of the RNA sequence of interest aregenerated.

[0019] In another aspect, the invention provides methods of generatingmultiple copies of the complementary sequence of an RNA sequence ofinterest comprising incubating a reaction mixture, said reaction mixturecomprising: (a) a single stranded second primer extension productresulting from step (d) above; (b) a third primer comprising a sequencehybridizable to an RNA transcript comprising a sequence complementary tothe target RNA; (c) a propromoter polynucleotide comprising apropromoter and a region which is hybridizable to a single strandedsecond primer extension product; (d) a propromoter polynucleotidecomprising a propromoter and a region which is hybridizable to a singlestranded third primer extension product; (e) an enzyme that cleaves RNAfrom an RNA/DNA hybrid; and (f) an RNA polymerase; wherein theincubation is under conditions that permit primer extension, RNAcleavage, propromoter polynucleotide hybridization and RNAtranscription, whereby multiple copies of the complementary sequence ofthe RNA sequence of interest are generated.

[0020] In another aspect, the invention provides methods of generatingmultiple copies of the complementary sequence of an RNA sequence ofinterest, said method comprising incubating a reaction mixture, saidreaction mixture comprising: (a) an RNA transcript from step (e) above,(b) a third primer comprising a sequence hybridizable to the RNAtranscript; (c) a propromoter polynucleotide comprising a propromoterand a region which is hybridizable to a single stranded third primerextension product; (d) an enzyme that cleaves RNA from an RNA/DNAhybrid; and (e) an RNA polymerase; wherein the incubation is underconditions that permit primer extension, RNA cleavage, propromoterpolynucleotide hybridization and RNA transcription, whereby multiplecopies of the complementary sequence of the RNA sequence of interest aregenerated.

[0021] In another aspect, the invention provides methods of generatingmultiple copies of the complementary sequence of an RNA sequence ofinterest, said method comprising incubating a reaction mixture, saidreaction mixture comprising: (a) a target RNA; (b) a first primercomprising a sequence that is hybridizable to the target RNA; (c) asecond primer comprising a sequence hybridizable to an extension productof the first primer; (d) a propromoter polynucleotide comprising apropromoter and a region which is hybridizable to a single strandedsecond primer extension product; (e) an RNA-dependent DNA polymerase;(f) a DNA-dependent DNA polymerase; (g) an RNA polymerase; and (h) anenzyme that cleaves RNA from an RNA/DNA hybrid; wherein the incubationis under conditions that permit primer hybridization, primer extension,RNA cleavage, propromoter polynucleotide hybridization, and RNAtranscription, whereby multiple copies of the complementary sequence ofthe RNA sequence of interest are generated.

[0022] In another aspect, invention provides methods of generatingmultiple copies of the complementary sequence of an RNA sequence ofinterest, said method comprising incubating a reaction mixture, saidreaction mixture comprising: (a) a target RNA; (b) a first primercomprising a sequence that is hybridizable to the target RNA; (c) asecond primer comprising a sequence hybridizable to an extension productof the first primer; (d) a third primer comprising a sequencehybridizable to an RNA transcript comprising a sequence complementary tothe target RNA; (e) a propromoter polynucleotide comprising apropromoter and a region which is hybridizable to a single strandedsecond primer extension product; (f) a propromoter polynucleotidecomprising a propromoter and a region which is hybridizable to a singlestranded third primer extension product; (g) an RNA-dependent DNApolymerase;(h) a DNA-dependent DNA polymerase; (i) an RNA polymerase;and (j) an enzyme that cleaves RNA from an RNA/DNA hybrid; wherein theincubation is conditions that permit primer hybridization, primerextension, RNA cleavage, propromoter polynucleotide hybridization, andRNA transcription, whereby multiple copies of the complementary sequenceof the RNA sequence of interest are generated.

[0023] In another aspect, the invention provides methods of generatingmultiple copies of the complementary sequence of an RNA sequence ofinterest, said method comprising: (a) hybridizing a composite primer toa single stranded second primer extension product resulting from step(d) above, wherein the composite primer comprises an RNA portion and a3′ DNA portion; (b) extending the composite primer with a DNA-dependentDNA polymerase, whereby a complex comprising a primer extension productand the second primer extension product is formed; (c) cleaving RNA inthe complex of step (b) with an enzyme that cleaves RNA from an RNA/DNAhybrid, such that another composite primer hybridizes to the secondprimer extension product and repeats primer extension by stranddisplacement, whereby multiple copies of the complement of the RNAsequence of interest are produced.

[0024] In another aspect, the invention provides methods of generatingmultiple copies of a polynucleotide sequence complementary to an RNAsequence of interest, said method comprising the steps of: (a) extendinga composite primer hybridized to a second primer extension product,wherein said primer extension product comprises a complement of a firstprimer extension product generated by extension of a first primerhybridized to template RNA by any of the methods described herein;whereby said first primer extension product is displaced, and wherebymultiple copies of a polynucleotide sequence complementary to the RNAsequence of interest are generated.

[0025] In another aspect, the invention provides methods of generatingmultiple copies of the complementary sequence of an RNA sequence ofinterest, said method comprising incubating a reaction mixture, saidreaction mixture comprising: (a) a single stranded second primerextension product resulting from step (d) above; (b) a composite primerwhich is hybridizable to the single stranded second primer extensionproduct, wherein the composite primer comprises an RNA portion and a 3′DNA portion; (c) DNA-dependent DNA polymerase; (d) an enzyme thatcleaves RNA from an RNA/DNA hybrid; wherein the incubation is made underconditions that permit composite primer primer hybridization, RNAcleavage, and displacement of the primer extension product from thecomplex of step (a) described above when its RNA is cleaved and acomposite primer binds to the primer extension product in the complex,whereby multiple copies of the complement of the RNA sequence ofinterest are produced.

[0026] In another aspect, the invention provides methods of generatingmultiple copies of an RNA sequence of interest, said method comprisingthe steps of: (a) extending a composite primer hybridized to a secondprimer extension product, wherein said primer extension productcomprises a complement of a first primer extension product generated byextension of a first primer hybridized to template RNA by any of themethods described herein; whereby said first primer extension product isdisplaced, (b) hybridizing the displaced first primer extension productwith a polynucleotide comprising a propromoter and a region which ishybridizable to the displaced first primer extension product underconditions which allow transcription to occur by RNA polymerase, suchthat RNA transcripts are produced comprising sequences complementary tothe displaced primer extension products, whereby multiple copies of theRNA sequence of interest are generated.

[0027] As is clear to one skilled in the art, reference to production ofcopies of an RNA or DNA sequence of interest or copies of apolynucleotide sequence complementary to an RNA or DNA sequence ofinterest refers to products that may contain, comprise or consist ofsuch sequences. As is evident to one skilled in the art, aspects thatrefer to combining and incubating the resultant mixture also encompassesmethod embodiments which comprise incubating the various mixtures (invarious combinations and/or subcombinations) so that the desiredproducts are formed. It is understood that any combination of theseincubation steps, and any single incubation step, to the extent that theincubation is performed as part of any of the methods described herein,fall within the scope of the invention. It is also understood thatmethods that comprise one or more incubation steps do not require aseparate combination step, as such combinations are implicit inincubating the reaction mixture(s)

[0028] Various embodiments of the primers are used in the methods of theinvention. For example, in some embodiments, the first primer comprisesa 5′ portion that is not hybridizable (under a given set of conditions)to a target ribonucleic acid. In some of these embodiments, the 5′portion comprises a sequence the complement of which is hybridizable bya propromoter polynucleotide under a given set of conditions. In oneexample, the presence of said 5′ portion in the first primer results ingeneration of a second primer extension product that is hybridizable(under a given set of conditions) by a propromoter polynucleotide. Inanother example, the presence of said 5′ portion in the first primerresults in generation of a third primer extension product that ishybridizable (under a given set of conditions) by a propromoterpolynucleotide. In some embodiments wherein a target RNA is mRNA, thefirst primer may comprise a poly-T sequence. In other embodiments, thesecond primer and the third primer are the same. In still anotherembodiment, the second primer and the third primer are different. In yetanother embodiment, the second primer and the third primer hybridize todifferent complementary sequences. In some embodiments, the secondand/or third primer comprises a sequence (for example, a 3′ sequence)that is a random sequence. In yet other embodiments, the second and/orthird primer is a random primer. In yet other embodiments, the thirdprimer is a composite primer. In some embodiments, the RNA portion of acomposite primer is 5′ with respect to the 3′ DNA portion. In stillother embodiments, the 5′ RNA portion is adjacent to the 3′ DNA portion.In other embodiments, the composite primer comprises the sequence of afirst primer.

[0029] The enzymes which may be used in the methods and compositions aredescribed herein. For example, the enzyme that cleaves RNA may be anRNase H, and the RNA-dependent DNA polymerase may be reversetranscriptase. The RNA-dependent DNA polymerase may comprise an RNase Henzyme activity. Similarly, a DNA polymerase may comprise bothRNA-dependent and DNA-dependent DNA polymerase enzyme activities. ADNA-dependent DNA polymerase and an enzyme that cleaves RNA may also bethe same enzyme. A DNA-dependent DNA polymerase, an RNA-dependent DNApolymerase, and the enzyme that cleaves RNA can also be the same enzyme.

[0030] In some embodiments, methods of the invention are used togenerate labeled polynucleotide products (generally DNA or RNAproducts). In some embodiments of methods for generating labeled DNAproducts, at least one type of dNTP used is a labeled dNTP. In someembodiments of methods for generating labeled RNA products, at least onetype of rNTP used is a labeled rNTP. In other embodiments of methods forgenerating labeled DNA products, a labeled composite primer is used.

[0031] In some embodiments, the methods of the invention employ apropromoter polynucleotide (for example, a PTO) that comprises a regionat the 3′ end which hybridizes to the second or third primer extensionproducts, whereby DNA polymerase extension of the extension productsproduces a double stranded promoter from which transcription occurs.

[0032] The methods are applicable to amplifying any RNA target,including, for example, mRNA and ribosomal RNA. One or more steps may becombined and/or performed sequentially (often in any order, as long asthe requisite product(s) are able to be formed). It is also evident, andis described herein, that the invention encompasses methods in which theinitial, or first, step is any of the steps described herein. Forexample, the methods of the invention do not require that the first stepbe production of the first primer extension product from the RNAtemplate. Methods of the invention encompass embodiments in which later,“downstream” steps are an initial step.

[0033] The invention also provides methods which employ (usually,analyze) the products of the amplification methods of the invention,such as sequencing, detection of sequence alteration(s) (e.g.,genotyping or nucleic acid mutation detection); determining presence orabsence of a sequence of interest; gene expression profiling;subtractive hybridization; preparation of a subtractive hybridizationprobe; differential amplification; preparation of libraries (includingcDNA and differential expression libraries); preparation of animmobilized nucleic acid (which can be a nucleic acid immobilized on amicroarray), and characterizing (including detecting and/or quantifying)amplified nucleic acid products generated by the methods of theinvention.

[0034] In one aspect, the invention provides methods of sequencing anRNA sequence of interest, said method comprising (a) amplifying a targetribonucleic acid containing the sequence of interest by the methodsdescribed herein in the presence of a mixture of rNTPs and rNTP analogssuch that transcription is terminated upon incorporation of an rNTPanalog; and (b) analyzing the amplification products to determinesequence.

[0035] In another aspect, the invention provides methods of sequencingan RNA sequence of interest, said method comprising (a) amplifying atarget ribonucleic acid containing the sequence of interest by themethods described herein, wherein RNA transcripts generated from thesecond primer extension product are amplified in the presence of amixture of rNTPs and rNTP analogs such that transcription is terminatedupon incorporation of an rNTP analog; and (b) analyzing theamplification products to determine sequence.

[0036] In some aspects, the invention provides methods of sequencing anRNA sequence of interest, said methods comprising amplifying a targetRNA containing the sequence of interest by the amplification methods ofthe invention in the presence of a mixture of dNTPs and dNTP analogs(which may be labeled or unlabeled), such that primer extension isterminated upon incorporation of a dNTP analog which may be labeled orunlabeled, and analyzing the amplification products to determinesequence.

[0037] In some aspects, the invention provides methods of detecting amutation (or, in some aspects, characterizing a sequence) in a targetribonucleic acid, comprising (a) amplifying the target ribonucleic acidby a method described herein; and (b) analyzing the amplificationproducts of the method for single stranded conformation, wherein adifference in conformation as compared to a reference single strandedpolynucleotide indicates a mutation in the target ribonucleic acid. Inother embodiments, the invention provides methods of detecting amutation (or, in some aspects, characterizing a sequence) in a targetribonucleic acid comprising analyzing amplification products of any ofthe methods described herein for single stranded conformation, wherein adifference in conformation as compared to a reference single strandedpolynucleotide indicates a mutation in the target ribonucleic acid (or,in some aspects, characterizes the target sequence).

[0038] In another aspect, the invention provides methods of producing anucleic acid immobilized to a substrate (which includes methods ofproducing a microarray), comprising (a) amplifying a target RNA by anyof the methods described herein; and (b) immobilizing the amplificationproducts on a substrate. The amplification products can be labeled orunlabeled. In other aspects, the invention provides methods of producinga microarray, comprising (a) amplifying a target RNA by an amplificationmethod described herein; and (b) immobilizing the amplification productson a substrate (which can be solid or semi-solid). In some embodiments,microarrays are produced by immobilizing amplification products onto asubstrate to make a microarray of amplification products. In otherembodiments, microarrays are produced by immobilizing amplificationproducts by any of the methods described herein onto a solid substrateto make a microarray of amplification products. The microarray cancomprise at least one amplification product immobilized on a solid orsemi-solid substrate fabricated from a material selected from the groupconsisting of paper, glass, ceramic, plastic, polypropylene, nylon,polyacrylamide, nitrocellulose, silicon an other metals, and opticalfiber. An amplification product can be immobilized on the solid orsemi-solid substrate in a two-dimensional configuration or athree-dimensional configuration comprising pins, rods, fibers, tapes,threads, beads, particles, microtiter wells, capillaries, and cylinders.

[0039] Any of the methods of the invention can be used to generatepolynucleotide (generally, RNA or DNA) products that are suitable forcharacterization of an RNA sequence of interest in a sample. In oneembodiment, the invention provides methods for characterizing (forexample, detecting and/or quantifying and/or determining presence orabsence of) an RNA sequence of interest comprising: (a) amplifying atarget RNA by any of the methods described herein; and (b) analyzing theamplification products. Step (b) of analyzing the amplification productscan be performed by any method known in the art or described herein, forexample by detecting and/or quantifying and/or determining present orabsence of amplification products that are hybridized to a probe. Theseamplification products may or may not be labeled. Any of the methods ofthe invention can be used to generate polynucleotide (generally, RNA orDNA) products that are labeled by incorporating labeled nucleotides intoappropriate step(s) of the methods. These labeled products areparticularly suitable for quantification and/or identification and/ordetermining presence or absence of by methods known in the art, whichinclude the use of arrays such as cDNA microarrays and oligonucleotidearrays. In one aspect, the invention provides a method of characterizingan RNA sequence of interest, comprising (a) amplifying a target RNA by amethod described herein to generate labeled products; and (b) analyzingthe labeled products. In some embodiments, the step of analyzing RNAproducts comprises determining amount of said products, whereby theamount of the RNA sequence of interest present in a sample isquantified. The polynucleotide products can be analyzed by, for example,contacting them with at least one probe. In some embodiments, the atleast one probe is provided as a microarray. The microarray can compriseat least one probe immobilized on a solid or semi-solid substratefabricated from a material selected from the group consisting of paper,glass, plastic, polypropylene, nylon, polyacrylamide, nitrocellulose,silicon, and optical fiber. A probe can be immobilized on the solid orsemi-solid substrate in a two-dimensional configuration or athree-dimensional configuration comprising pins, rods, fibers, tapes,threads, beads, particles, microtiter wells, capillaries, and cylinders.

[0040] In another aspect, the invention provides methods of determininggene expression profile in a sample, the methods comprising: (a)amplifying at least one RNA sequence of interest in the sample using anyof the methods described herein; and (b) determining amount ofamplification products of each RNA sequence of interest, wherein eachsaid amount is indicative of amount of each RNA sequence of interest inthe sample, whereby the gene expression profile of the sample isdetermined.

[0041] In another aspect, the invention provides methods of preparing asubtractive hybridization probe, said methods comprising generatingmultiple single stranded polynucleotide, preferably DNA, copies of thecomplement of at least one RNA sequences of interest from a first RNApopulation using any of the methods described herein.

[0042] In another aspect, the invention provides methods of performingsubtractive hybridization, said methods comprising: (a) generatingmultiple copies of the complement of at least one RNA sequence ofinterest from a first RNA population using any of the methods describedherein; and (b) hybridizing the multiple copies to a second mRNApopulation, whereby a subpopulation of the second mRNA population formsa complex with a copy of the complement of at least one RNA sequence ofinterest. In embodiments in which DNA copies are generated, the methodsfurther comprise: (c) cleaving RNA in the complex of step (b) with anenzyme that cleaves RNA from an RNA/DNA hybrid; and (d) amplifying anunhybridized subpopulation of the second mRNA population (using anymethod, including the methods described herein), whereby multiple copiesof single stranded DNA complementary to the unhybridized subpopulationof the second mRNA population are generated.

[0043] In another aspect, the invention provides methods fordifferential amplification, the methods comprising: (a) generatingmultiple nucleic acid (generally DNA) copies of the complement of atleast one RNA sequence of interest from a first RNA population using anyof the methods described herein; (b) hybridizing the multiple copies toa second mRNA population, whereby a subpopulation of the second mRNApopulation forms a complex with a DNA copy; (c) cleaving RNA in thecomplex of step (b) with an enzyme that cleaves RNA from an RNA/DNAhybrid; and (d) amplifying an unhybridized subpopulation of the secondmRNA population using any method, including those described herein,whereby multiple copies of single stranded DNA complementary to theunhybridized subpopulation of the second mRNA population are generated.These methods encompass steps (b), (c) and (d) if the copies used in thesubtractive hybridization are generated using any of the methodsdescribed herein.

[0044] In another aspect, the invention provides methods for making alibrary, said method comprising preparing a subtractive hybridizationprobe as described herein, or differential amplification as describedherein. Any of these applications can use any of the amplificationmethods (including various components and various embodiments of any ofthe components) as described herein.

[0045] The invention also provides compositions, kits, complexes,reaction mixtures and systems comprising various components (and variouscombinations of the components) used in the amplification methodsdescribed herein. The compositions may be any component(s), reactionmixture and/or intermediate described herein, as well as any combinationthereof.

[0046] In some embodiments, the invention provides a compositioncomprising: (a) a first primer (which can be a random primer); (b) asecond primer (which can be a random primer); and (c) a propromoterpolynucleotide (which in some embodiments is a PTO). In someembodiments, these compositions may further comprise: (d) a third primer(which can be a random primer). In some of these embodiments, the firstprimer comprises a sequence that is not hybridizable to a target RNA. Insome of these embodiments, the second primer comprises a sequence thatis not hybridizable to a first primer extension product. In someembodiments, the third primer comprises a sequence that is nothybridizable to an RNA transcript. In some embodiments, the propromoterpolynucleotide ((c), above) is capable of hybridizing to the complementof the 5′ portion of the first primer.

[0047] The invention also provides compositions comprising a propromoterpolynucleotide (such as a PTO) capable of hybridizing to a 3′ portion ofa second primer extension that is complement of a 5′ portion of a firstprimer used to create first primer extension product.

[0048] The invention also provides compositions comprising (a) a firstprimer; (b) a second primer (which can be a random primer); and (c) acomposite primer, wherein the composite primer comprises a 5′ RNAportion and a DNA portion. In some embodiments, the invention provides acomposition comprising: (a) a first primer (which can be a randomprimer) hybridizable to target RNA; (b) a second primer (which can be arandom primer); and (c) a composite primer hybridizable to a secondprimer extension product. In some embodiments, the composition furthercomprises one or more of the following: DNA-dependent DNA polymerase,RNA-dependent DNA polymerase, and an agent (generally an enzyme) thatcleaves RNA from an RNA/DNA heteroduplex.

[0049] The invention also provides compositions comprising theamplification products described herein. Accordingly, the inventionprovides a population of anti-sense RNA molecules which are copies of atarget sequence, which are produced by any of the methods describedherein. The invention also provides a population of anti-sensepolynucleotides (generally DNA) molecules, which are produced by any ofthe methods described herein.

[0050] In another aspect, the invention provides compositions comprisingany of the complexes (which are generally considered as intermediateswith respect to the final amplification products) described herein (seealso the figures for schematic depictions of examples of these variouscomplexes). For example, the invention provides compositions comprisinga complex of (a) a first primer extension product; and (b) a target RNAstrand. In yet another aspect, the invention provides compositionscomprising a complex of: (a) a first primer extension product; and (b) asecond primer extension product. In another example, the inventionprovides compositions comprising a complex of (a) a second primerextension product; and (b) a propromoter polynucleotide (which can be aPTO). In some embodiments, the propromoter polynucleotide hybridizes toa sequence in the second primer extension product comprising thecomplement of the 5′ portion of a first prime, wherein the first primeris extended to form the first primer extension product. In yet anotherexample, the invention provides compositions comprising a complex of (a)a third primer extension product; and (b) a propromoter polynucleotide(which can be a PTO). In yet another example, the invention providescompositions comprising a complex of (a) a second primer extensionproduct, generated by denaturation of a hybridized first and secondprimer extension product; and (b) a composite primer hybridizable to thesecond primer extension product.

[0051] In another aspect, the invention includes any one or moreproducts (including intermediates) and compositions comprising theproducts (including intermediates) produced by any aspect of the methodsof the invention. The products include libraries and any otherpopulation produced, which are generally based on the nature of theprimer(s) used in the methods described herein.

[0052] In another aspect, the invention provides reaction mixtures (orcompositions comprising reaction mixtures) which contain variouscombinations of components described herein. For example, the inventionprovides reaction mixtures comprising (a) a target RNA; (b) a firstprimer; (c) a second primer (which can be a random primer); (d) an RNApolymerase; and (e) a DNA polymerase. The reaction mixture could alsofurther comprise an enzyme which cleaves RNA from an RNA/DNA hybrid,such as RNase H. A reaction mixture of the invention can also comprise apropromoter polynucleotide (which in some embodiments is a PTO).

[0053] In another aspect, the invention provides reaction mixturescomprising (a) a target RNA; (b) a first primer; (c) a second primer(which can be a random primer); (d) an RNA polymerase; (e) a DNApolymerase; and (f) a composite primer. The reaction mixture could alsofurther comprise an enzyme which cleaves RNA from an RNA/DNA hybrid,such as RNase H.

[0054] In another aspect, the invention provides kits for conducting themethods described herein. These kits, in suitable packaging andgenerally (but not necessarily) containing suitable instructions forperforming any of the methods of the invention described herein,including sequencing, detection of sequence alteration(s) (e.g.,genotyping or nucleic acid mutation detection); determining presence orabsence of a sequence of interest; gene expression profiling;subtractive hybridization; preparation of a subtractive hybridizationprobe; differential amplification; preparation of libraries (includingcDNA and differential expression libraries); preparation of animmobilized nucleic acid (which can be a nucleic acid immobilized on amicroarray), and characterizing (including detecting and/or quantifyingand/or determining presence or absence of) amplified nucleic acidproducts generated by the methods of the invention. The kits furthercomprise one or more components used in the methods of the invention.For example, the invention provides kits that comprise a first primerthat comprises a sequence the complement of which is hybridizable by apropromoter polynucleotide, and instructions for using the primer toamplify RNA. The invention also provides kits that further comprise asecond primer and/or a third primer, and optionally instructions forusing the primers to amplify RNA. The kits can contain furthercomponents, such as any of (a) a propromoter polynucleotide (such as aPTO); and (b) any of the enzymes described herein, such as an enzymewhich cleaves RNA from an RNA/DNA hybrid (for example, RNaseH), DNApolymerase (RNA-dependent or DNA-dependent) and RNA polymerase. Inanother example, a kit can comprise (a) a composite primer; and (b)instructions for using the composite primer to amplify target RNA usingthe methods of the invention provided herein. The kit can comprisefurther components, including any of the enzymes described herein, suchas an enzyme which cleaves RNA from an RNA/DNA hybrid (for example,RNaseH), and DNA polymerase (RNA-dependent or DNA-dependent). In anotherexample, a kit comprises a first primer that comprises a sequence thecomplement of which is hybridizable by a propromoter polynucleotide, andinstructions for using the primer to amplify RNA using any of themethods described herein. In another embodiment, the kit furthercomprises a second primer.

[0055] In another aspect, the invention provides systems for effectingthe amplification methods described herein. For example, the inventionprovides systems for amplifying a target ribonucleic acid, comprising:(a) a first primer; (b) a second primer (which can be a random primer);(c) an RNA-dependent DNA polymerase; (d) a DNA-dependent DNA polymerase;(e) a propromoter polynucleotide (such as a PTO); and (f) an enzymewhich cleaves RNA from an RNA/DNA hybrid (such as RNaseH). The systemsmay also comprise: (g) a third primer (which can be a random primer). Asdescribed herein, systems of the invention generally comprise one ormore apparatuses appropriate for carrying out methods of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0056]FIG. 1 is a diagrammatic representation of a linear RNAamplification process. FIG. 1 shows amplification of a target RNA usinga polynucleotide comprising a propromoter to produces multiple copies ofRNA transcripts complementary to the target RNA.

[0057] FIGS. 2A-B show further amplification of the RNA transcripts fromthe process of FIG. 1 to generate more RNA transcripts complementary tothe target RNA.

[0058] FIGS. 3A-B show a diagrammatic representation of a linear RNAamplification process using a third primer that is a composite primer togenerate single stranded DNA strands complementary to the target RNA.

MODES FOR CARRYING OUT THE INVENTION

[0059] The invention provides methods, compositions and kits foramplifying polynucleotide sequences, specifically ribonucleic acid (RNA)sequences. The methods provide for amplification of a single RNA speciesor pool of RNA species. The methods can achieve exponentialamplification, which would be particularly useful for amplification ofvery low amounts of RNA sequences in a biological sample. The methodsare suitable for, for example, generation of libraries, including cDNAlibraries. They generate single stranded RNA or, in some embodiments,single stranded DNA products, which are readily suitable for multiplexanalysis by microarray technologies, as well as electrophoresis-basedtechnologies such as differential display. The methods are amenable toautomation and do not require thermal cycling. The methods generallycomprise hybridizing a polynucleotide comprising a propromoter sequenceto a primer extension product to generate an intermediate productcapable of driving transcription, whereby RNA transcripts comprisingsequences complementary to an RNA sequence of interest are produced. Inanother aspect, the methods comprise isothermal linear amplification ofDNA copies complementary to the RNA sequence of interest using adenaturation step, a composite primer and strand displacement. See Kurn,U.S. Pat. No. 6,251,639 B1.

[0060] The methods of the invention are directed to the amplification ofone or more species of RNA, such as a pool of RNA sequences, and is mostparticularly suitable for the amplification of all RNA (such as mRNA)sequences in a preparation of total RNA from a biological sample. Thus,one of the major advantages of the methods of the invention is theability to amplify an entire pool of sequences, which is essential forthe ability to analyze the gene expression profile in cells, such as thecells in a biological of interest sample. The methods of the inventionhave the potential of amplifying a multiplicity, more preferably a largemultiplicity, and most preferably all RNA (such as mRNA) sequences in asample.

[0061] Insofar as many mRNAs have a unique polyA 3′-end, theamplification initiated from the 3′-end sequence of mRNAs is most commonfor preparation of cDNA libraries and subsequent sequence analysis fordetermination of gene expression profiling or other applications. Themethods of the invention are similarly suited for preparation oflibraries of amplified 3′-portions of mRNAs. The sequence of the firstprimer used in the methods of invention can be designed to becomplementary to a multiplicity, or all, of the mRNA species in thesample by using random sequences, according to methods known in the art.

[0062] Various methods for mRNA amplification have been described. U.S.Pat. Nos., 744,308; 6,143,495; EP 0971039A 2; EP 0878553A2. Most ofthese methods are transcription based, wherein a promoter for RNApolymerase is incorporated into a double stranded cDNA by a primercomprising a propromoter sequence at the 5′-end which hybridizes totarget RNA. These primers can non-specifically bind to template RNA.Insofar as a DNA polymerase has a high affinity for primer hybridized toa template nucleic acid with a free 3′ end, i.e. a substrate for primerextension by the polymerase, it is highly probable that a primercomprising a propromoter sequence at the 5′ end may non-specificallyincorporate the promoter sequence into an amplification product. Thisresults in uncontrolled production of transcription products. Theappending of a double stranded promoter by a propromoter polynucleotide,as described herein, provides for increased specificity and control ofthe transcription-based generation of amplification product.

[0063] In one aspect, the invention works as follows: generation ofmultiple copies of the complementary sequence of an RNA sequence ofinterest is achieved by using a first primer (which can be a specific orrandom primer) that comprises a sequence (generally, in its 5′ portion)the complement of which is hybridizable by a polynucleotide comprising apropromoter. In some embodiments, the sequence the complement of whichis hybridizable by a polynucleotide comprising a propromoter is asequence that is hybridizable to a target RNA when the primer ishybridized to the target RNA. In other embodiments, the sequence thecomplement of which is hybridizable by a polynucleotide comprising apropromoter is a sequence that is not hybridizable to a target RNA whenthe primer is hybridized to the target RNA (thus forming a tail when theprimer is hybridized to a target). The extension of the first primeralong a target RNA by an RNA-dependent DNA polymerase results in thegeneration of an intermediate polynucleotide (first primer extensionproduct) that has at least one defined end (the first primer end). Aftercleavage of the template RNA from a complex comprising the target RNAand first primer extension product, a second primer (which can be aspecific or random primer) is then hybridized to the first DNA strand(first primer extension product) and extended to form a complex of firstand second primer extension products that at one end comprises asequence to which a polynucleotide comprising a propromoter ishybridizable. The second primer is any sequence that is hybridized tothe first DNA strand such that it is capable of being extended by a DNApolymerase along a first DNA strand to generate a second DNA strand.Thus, in some embodiments, the second primer is an oligonucleotide (thatis separately provided). In other embodiments, it is or comprises asequence of the first DNA strand (generally at the 3′ end) that ishybridized to a sequence in the first DNA strand (for example, a hairpinor self-annealed structure). Following denaturation of the complex, apolynucleotide comprising a propromoter is hybridized to the secondprimer extension product and the 3′ end of the primer extension productis extended along the propromoter oligonucleotide (if there is anyoverhang) to generate a double stranded promoter region. RNAtranscription driven by this promoter results in generation of multiplecopies of RNA transcripts comprising the complementary sequence of theRNA sequence of interest. In some embodiments involving cyclicalamplification (also referred to herein as “exponential” amplification),these RNA transcripts, and optionally RNA transcripts generated insubsequent steps, are hybridized with a third primer (which may or maynot be the same as the second primer, and which may be a specific orrandom primer). The third primer is extended to form a complexcomprising the RNA transcript and a third primer extension product(which constitutes a DNA/RNA heteroduplex). Cleavage of the RNAtranscript results in a single stranded third primer extension productto which a propromoter polynucleotide hybridizes. If necessary (i.e., ifthere is an overhang), the third primer extension product is extendedalong the propromoter polynucleotide to generate a double strandedpromoter region. RNA transcription driven by this promoter results ingeneration of multiple copies of RNA transcripts comprising thecomplementary sequences of the RNA sequence of interest. Hybridizationof third primers to these RNA transcripts initiates a cyclical processleading to further amplification.

[0064] Accordingly, the invention provides methods of producing at leastone copy of the complementary sequence of an RNA sequence of interest,said method comprising combining and reacting the following: (a) atarget RNA comprising an RNA sequence of interest; (b) a first primerthat hybridizes to the target RNA; (c) a second primer that ishybridizable to an extension product of the first primer; (d) anRNA-dependent DNA polymerase; (e) a DNA-dependent DNA polymerase; (f) anenzyme that cleaves RNA from an RNA/DNA hybrid; (g) a propromoterpolynucleotide comprising a propromoter and a region which hybridizes toan extension product of the second primer; (h) deoxyribonucleosidetriphosphates or suitable analogs; (i) ribonucleoside triphosphates andsuitable analogs; and (j) an RNA polymerase. In embodiments involvingcyclical amplification (interchangeably termed “exponential”amplification, herein), the following are also included in theamplification reaction (either at the same time as the components listedabove or added separately): (k) optionally a third primer (which may ormay not be the same as the second primer) that hybridizes to an RNAtranscript comprising sequences complementary to the sequence of thetarget RNA; and (l) optionally a second polynucleotide comprising apropromoter and a region which hybridizes to a single stranded thirdprimer extension product (this polynucleotide may or may not be the sameas the polynucleotide described in (g) above).

[0065] As is evident to one skilled in the art, by this disclosure, thereactions described may be simultaneous or sequential, as such, theinvention includes these various embodiments and combinations.

[0066] In some embodiments, the invention provides methods of producingat least one copy of the complementary sequence of an RNA sequence ofinterest, said method comprising combining and reacting the following:(a) a single stranded second primer extension product resulting fromdenaturation of a complex of first and second primer extension productsas described herein; (b) a propromoter polynucleotide comprising apropromoter and a region which hybridizes to a second primer extensionproduct; (c) ribonucleoside triphosphates and suitable analogs; and (d)an RNA polymerase. In embodiments involving cyclical amplification(“exponential” amplification), the following may also included in theamplification reaction (either at the same time as the components listedabove or added separately): (e) optionally a third primer (which may ormay not be the same as the second primer) that hybridizes to an RNAtranscript comprising sequences complementary to the sequence of thetarget RNA; and (f) optionally a second polynucleotide comprising apropromoter and a region which hybridizes to a single stranded thirdprimer extension product (this polynucleotide may or may not be the sameas the polynucleotide described in (b) above). In some embodimentsinvolving cyclical amplification, said method comprises combining andreacting (under suitable conditions and reagent such that multiplecopies of a polynucleotide sequence complementary to an RNA sequence ofinterest are produced) the following: (a) an RNA transcript generatedfrom the complex of the first propromoter polynucleotide and secondprimer extension product; (b) a third primer (which may or may not bethe same as the second primer) that hybridizes to the RNA transcript;(c) a second polynucleotide comprising a propromoter and a region whichhybridizes to a single stranded third primer extension product (thispolynucleotide may or may not be the same as the propromoterpolynucleotide in the complex from which the RNA transcript in step (a)is generated).

[0067] In another aspect, the invention provides a method of generatingmultiple copies of a polynucleotide sequence complementary to an RNAsequence of interest as follows: generation of multiple copies of thecomplementary sequence of an RNA sequence of interest is achieved byusing a first primer (which can be a specific or random primer) thatcomprises a sequence in its 5′ portion that is not hybridizable totarget RNA (thus forming a tail when the primer is hybridized to atarget under conditions when the first primer hybridizes to templateRNA). The extension of the first primer along a target RNA by anRNA-dependent DNA polymerase results in the generation of anintermediate polynucleotide (first strand cDNA) that has at least onedefined end comprising the complement of the first primer sequence.After cleavage of the template RNA from a complex comprising the targetRNA and first strand cDNA, a second primer (which can be a specific orrandom primer) is then hybridized to the first strand cDNA) and extendedto form a complex of first and second strand cDNAs that at (at least)one end has a defined end comprising the first primer sequence and thecomplement of the first primer sequence. The second primer is anysequence that is hybridized to the first DNA strand such that it iscapable of being extended by a DNA polymerase along a first DNA strandto generate a second DNA strand. Thus, in some embodiments, the secondprimer is an oligonucleotide (that is separately provided). In otherembodiments, it comprises a sequence of the first DNA strand (generallyat the 3′ end) that is hybridized to a sequence in the first DNA strand(for example, a hairpin or self-annealed structure). In otherembodiment, the second primer comprises one or more fragments of thetarget RNA sequence that remains hybridized to the first primerextension product (after cleavage of the initial complex comprising thetarget RNA and first primer extension product). The complex of first andsecond strand cDNAs (wherein the second strand cDNA comprises a 3′ endportion that is the complement of the first primer sequence) is thendenatured to form single stranded first strand cDNA and second strandcDNA.

[0068] The single stranded second strand cDNA is the substrate forisothermal linear amplification using a composite primer and stranddisplacement as follows: a composite primer comprising a 5′-RNA portionand a DNA portion hybridizes to the 3′-portion of the second strandcDNA, generally to the 3′-portion of the second strand cDNA, and isextended along the second strand cDNA by a DNA polymerase to form adouble stranded complex comprising an RNA/DNA hybrid portion at one endof the complex. An enzyme which cleaves RNA from an RNA/DNA hybrid (suchas RNase H) cleaves RNA sequence from the hybrid, leaving a sequence onthe second strand cDNA available for binding by another compositeprimer. Another first (composite) strand cDNA is produced by DNApolymerase, which displaces the previously bound cleaved first strandcDNA, resulting in displaced cleaved first strand cDNA. The displacedcleaved first strand cDNA product comprises single stranded DNAcomplementary to the RNA sequence of interest. Kum, U.S. Pat. No.6,251,639 B1.

[0069] Any of the methods of the invention can be used to generatepolynucleotide (generally, RNA or DNA) products that are labeled byincorporating labeled nucleotides into appropriate steps of the methods.These labeled products are particularly suitable for quantificationand/or identification by methods known in the art, which include the useof arrays such as cDNA microarrays and oligonucleotide arrays.

[0070] In some embodiments, the invention provides methods of sequencingRNA sequences. For sequencing methods based on methods described herein,the appropriate rNTPs, or analogs thereof, which may be labeled orunlabeled, are used. Accordingly, the invention provides methods ofsequencing a target RNA comprising a sequence of interest based on themethods described above, wherein rNTPs and rNTP analogs which are primerelongation terminators, which may be labeled or unlabeled, are used, andthe amplification product is analyzed for sequence information, asdescribed below. For sequencing methods based on methods describedherein wherein the amplified product is DNA, the appropriate dNTPs, oranalogs thereof, which may be labeled or unlabeled, are used.

[0071] In other embodiments, the invention provides methods of detectingnucleic acid sequence mutations. In one embodiment, the amplificationproducts are used to detect and/or identify single strand conformationpolymorphisms in a target RNA.

[0072] The invention provides methods to characterize (for example,detect and/or quantify and/or determine presence or absence of ) an RNAsequence of interest by generating polynucleotide (generally RNA or DNA)products using amplification methods of the invention, and analyzing theproducts by detection/quantification methods such as those based onarray technologies or solution phase technologies. Generally, but notnecessarily, these amplified products are labeled.

[0073] In another aspect, the invention provides a method ofcharacterizing an RNA sequence of interest, comprising (a) amplifying atarget RNA by a method described herein to generate labeled products;and (b) analyzing the labeled polynucleotide (generally, RNA or DNA)products. In some embodiments, the step of analyzing products comprisesdetermining amount of said products, whereby the amount of the RNAsequence of interest present in a sample is quantified. Thepolynucleotide (generally, DNA or RNA) products can be analyzed by, forexample, contacting them with at least one probe. In some embodiments,the at least one probe is provided as a microarray. The microarray cancomprise at least one probe immobilized on a solid or semi-solidsubstrate fabricated from a material selected from the group consistingof paper, glass, ceramics, plastic, polypropylene, polystyrene, nylon,polyacrylamide, nitrocellulose, silicon, other metals, and opticalfiber. A probe can be immobilized on the solid or semi-solid substratein a two-dimensional configuration or a three-dimensional configurationcomprising pins, rods, fibers, tapes, threads, beads, particles,microtiter wells, capillaries, and cylinders.

[0074] In another aspect, the invention provides methods of determininggene expression profile in a sample, the methods comprising: (a)amplifying at least one RNA sequence of interest in the sample using anyof the methods described herein; and (b) determining amount ofamplification products of each RNA sequence of interest, wherein eachsaid amount is indicative of amount of each RNA sequence of interest inthe sample, whereby the gene expression profile of the sample isdetermined.

[0075] In another embodiment, the invention provides methods ofgenerating libraries (including cDNA libraries and subtractivehybridization libraries), said methods comprising: amplifying at leastone RNA sequences of interest using any of the methods described hereinto generate single stranded nucleic acid product, and methods ofgenerating and using subtractive hybridization probes, methods forsubtractive hybridization, methods for differential amplification, andmethods of generating subtractive hybridization libraries.

[0076] Various methods for the detection and quantification of geneexpression levels have been developed in recent years. For example,microarrays, in which either defined cDNAs or oligonucleotides areimmobilized at discrete locations on, for example, solid or semi-solidsubstrates, or on defined particles, enables the detection and/orquantification of the expression of a multitude of genes in a givenspecimen.

[0077] Using these previously known methods to detect and/or quantifymultiple mRNA species in a sample, which in turn is a measure of geneexpression profiling, generally requires direct labeling of cDNA, whichrequires a large amount of input total RNA, in part because mRNArepresents only a small subset of the total RNA pool. Thus, when usingtotal RNA preparations from a given cell or tissue sample, the analysisof gene expression in the sample using methods such as arrays requires asubstantial amount of input RNA, which generally ranges from 50 to 200μg.

[0078] Similarly, 2 to 5 μg of mRNA purified from a total RNApreparation would generally be required for a single analysis of geneexpression profiling using array technologies. This is a clearlimitation of methods such as those based on array technology, insofaras the number of cells, or size of tissue specimen required is verylarge, and these cells or tissue specimens are often scarcely availablefor testing or are too precious. This limitation is especially severe inthe study of clinical specimens, where the cells to be studied are rareand/or difficult to cultivate in vitro, and in high throughput screeningof libraries of effector molecules.

General Techniques

[0079] The practice of the invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel etal., eds., 1987, and periodic updates); “PCR: The Polymerase ChainReaction”, (Mullis et al., eds., 1994).

[0080] Primers, oligonucleotides and polynucleotides employed in theinvention can be generated using standard techniques known in the art.

Definitions

[0081] A “target sequence,” “target nucleic acid,” or “target RNA,” asused herein, is a polynucleotide comprising a sequence of interest, forwhich amplification is desired. The target sequence may be known or notknown, in terms of its actual sequence. In some instances, the terms“target” and “template”, and variations thereof, are usedinterchangeably.

[0082] “Amplification,” as used herein, generally refers to the processof producing multiple copies of a desired sequence. “Multiple copies”mean at least 2 copies. A “copy” does not necessarily mean perfectsequence complementarity or identity to the template sequence. Forexample, copies can include nucleotide analogs such as deoxyinosine,intentional sequence alterations (such as sequence alterationsintroduced through a primer comprising a sequence that is hybridizable,but not complementary, to the template, or a non-target sequenceintroduced through a primer), and/or sequence errors that occur duringamplification. “Amplifying” a sequence may generally refer to makingcopies of a sequence or its complement, with the understanding that, asstated above, copying does not necessarily mean perfect sequencecomplementarity or identity with respect to the template sequence.

[0083] “Polynucleotide,” or “nucleic acid,” as used interchangeablyherein, refer to polymers of nucleotides of any length, and include DNAand RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides,modified nucleotides or bases, and/or their analogs, or any substratethat can be incorporated into a polymer by DNA or RNA polymerase. Apolynucleotide may comprise modified nucleotides, such as methylatednucleotides and their analogs. If present, modification to thenucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.Other types of modifications include, for example, “caps”, substitutionof one or more of the naturally occurring nucleotides with an analog,internucleotide modifications such as, for example, those with unchargedlinkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates,cabamates, etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine,psoralen, etc.), those containing chelators (e.g., metals, radioactivemetals, boron, oxidative metals, etc.), those containing alkylators,those with modified linkages (e.g., alpha anomeric nucleic acids, etc.),as well as unmodified forms of the polynucleotide(s). Further, any ofthe hydroxyl groups ordinarily present in the sugars may be replaced,for example, by phosphonate groups, phosphate groups, protected bystandard protecting groups, or activated to prepare additional linkagesto additional nucleotides, or may be conjugated to solid supports. The5′ and 3′ terminal OH can be phosphorylated or substituted with aminesor organic capping groups moieties of from 1 to 20 carbon atoms. Otherhydroxyls may also be derivatized to standard protecting groups.Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including, forexample, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro- or 2′-azido-ribose,carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such asarabinose, xyloses or lyxoses, pyranose sugars, furanose sugars,sedoheptuloses, acyclic analogs and abasic nucleoside analogs such asmethyl riboside. One or more phosphodiester linkages may be replaced byalternative linking groups. These alternative linking groups include,but are not limited to, embodiments wherein phosphate is replaced byP(O)S(“thioate”), P(S)S (“dithioate”), “(O)NR₂ (“amidate”), P(O)R,P(O)OR′, CO or CH₂ (“formacetal”), in which each R or R′ isindependently H or substituted or unsubstituted alkyl (1-20 C)optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl,cycloalkenyl or araldyl. Not all linkages in a polynucleotide need beidentical. The preceding description applies to all polynucleotidesreferred to herein, including RNA and DNA.

[0084] A “labeled dNTP,” or “labeled rNTP,” as used herein, refers,respectively, to a dNTP or rNTP, or analogs thereof, that is directly orindirectly attached with a label. For example, a “labeled” dNTP or rNTP,may be directly labeled with, for example, a dye and/or a detectablemoiety, such as a member of a specific binding pair (such asbiotin-avidin). A “labeled” dNTP or rNTP, may also be indirectly labeledby its attachment to, for example, a moiety to which a label is/can beattached. A dNTP or rNTP, may comprise a moiety (for example, an aminegroup) to which a label may be attached following incorporation of thedNTP or rNTP into an extension product. Useful labels in the presentinvention include fluorescent dyes (e.g., fluorescein isothiocyanate,Texas red, rhodamine, green fluorescent protein and the like),radioisotopes (e.g., ³H, ³⁵S, ³²P, ³³P, ¹²⁵I, or ¹⁴C), enzymes (e.g.,LacZ, horseradish peroxidase, alkaline phosphatase, ), digoxigenin, andcolorimetric labels such as colloidal gold or colored glass or plastic(e.g., polystyrene, polypropylene, latex, etc.) beads. Variousanti-ligands and ligands can be used (as labels themselves or as a meansfor attaching a label). In the case of a ligand that has a naturalanti-ligand, such as biotin, thyroxine and cortisol, the ligand can beused in conjunction with labeled anti-ligands.

[0085] The “type” of dNTP or rNTP, as used herein, refers to theparticular base of a nucleotide, namely adenine, cytosine, thymine,uridine, or guanine.

[0086] “Oligonucleotide,” as used herein, generally refers to short,generally single stranded, generally synthetic polynucleotides that aregenerally, but not necessarily, less than about 200 nucleotides inlength. Oligonucleotides in the invention include the first, second, andthird primers, and propromoter polynucleotide (such as the PTO). Theterms “oligonucleotide” and “polynucleotide” are not mutually exclusive.The description above for polynucleotides is equally and fullyapplicable to oligonucleotides.

[0087] A “primer” is generally a nucleotide sequence (i.e. apolynucleotide), generally with a free 3′-OH group, that hybridizes witha template sequence (such as a target RNA, or a primer extensionproduct) and is capable of promoting polymerization of a polynucleotidecomplementary to the template. A “primer” can be, for example, anoligonucleotide. It can also be, for example, a sequence of the template(such as a primer extension product or a fragment of the templatecreated following RNase cleavage of a template-DNA complex) that ishybridized to a sequence in the template itself (for example, as ahairpin loop), and that is capable of promoting nucleotidepolymerization. Thus, a primer can be an exogenous (e.g., added) primeror an endogenous (e.g., template fragment) primer.

[0088] A “random primer,” as used herein, is a primer that comprises asequence that is designed not necessarily based on a particular orspecific sequence in a sample, but rather is based on a statisticalexpectation (or an empirical observation) that the sequence of therandom primer is hybridizable (under a given set of conditions) to oneor more sequences in the sample. The sequence of a random primer (or itscomplement) may or may not be naturally-occurring, or may or may not bepresent in a pool of sequences in a sample of interest. Theamplification of a plurality of RNA species in a single reaction mixturewould generally, but not necessarily, employ a multiplicity, preferablya large multiplicity, of random primers. As is well understood in theart, a “random primer” can also refer to a primer that is a member of apopulation of primers (a plurality of random primers) which collectivelyare designed to hybridize to a desired and/or a significant number oftarget sequences. A random primer may hybridize at a plurality of siteson a nucleic acid sequence. The use of random primers provides a methodfor generating primer extension products complementary to a targetpolynucleotide which does not require prior knowledge of the exactsequence of the target.

[0089] “Protopromoter sequence,” and “propromoter sequence,” as usedherein, refer to a single-stranded DNA sequence region which, indouble-stranded form is capable of mediating RNA transcription. In somecontexts, “protopromoter sequence,” “protopromoter,” “propromotersequence,” “propromoter,” “promoter sequence,” and “promoter” are usedinterchangeably.

[0090] A “propromoter polynucleotide,” as used herein, refers to apolynucleotide comprising a propromoter sequence. Example of apropromoter polynucleotide is a propromoter template oligonucleotide(PTO).

[0091] “Propromoter template oligonucleotide (PTO)” and “promotertemplate oligionucleotide (PTO)” as used herein, refer to anoligonucleotide that comprises a propromoter sequence and a portion,generally a 3′ portion, that is hybridizable (under a given set ofconditions) to the 3′ region of a primer extension product. Thepropromoter sequence and the hybridizable portion may be the same,distinct or overlapping nucleotides of an oligonucleotide.

[0092] To “inhibit” is to decrease or reduce an activity, function,and/or amount as compared to a reference.

[0093] A “complex” is an assembly of components. A complex may or maynot be stable and may be directly or indirectly detected. For example,as is described herein, given certain components of a reaction, and thetype of product(s) of the reaction, existence of a complex can beinferred. For purposes of this invention, a complex is generally anintermediate with respect to the final amplification product(s). Anexample of a complex is a nucleic acid duplex comprising a first primerextension product and a second primer extension product.

[0094] “Denaturing,” or “denaturation of” a complex comprising twopolynucleotides (such as a first primer extension product and a secondprimer extension product) refers to dissociation of two hybridizedpolynucleotide sequences in the complex. The dissociation may involve aportion or the whole of each polynucleotide. Thus, denaturing ordenaturation of a complex comprising two polynucleotides can result incomplete dissociation (thus generating two single strandedpolynucleotides), or partial dissociation (thus generating a mixture ofsingle stranded and hybridized portions in a previously double strandedregion of the complex).

[0095] A “portion” or “region,” used interchangeably herein, of apolynucleotide or oligonucleotide is a contiguous sequence of 2 or morebases. In other embodiments, a region or portion is at least about anyof 3, 5, 10, 15, 20, 25 contiguous nucleotides.

[0096] A region, portion, or sequence which is “adjacent” to anothersequence directly abuts that region, portion, or sequence.

[0097] A “reaction mixture” is an assemblage of components, which, undersuitable conditions, react to form a complex (which may be anintermediate) and/or a product(s).

[0098] “A”, “an” and “the”, and the like, unless otherwise indicatedinclude plural forms.

[0099] “Comprising” means including.

[0100] Conditions that “allow” an event to occur or conditions that are“suitable” for an event to occur, such as hybridization, strandextension, and the like, or “suitable” conditions are conditions that donot prevent such events from occurring. Thus, these conditions permit,enhance, facilitate, and/or are conducive to the event. Such conditions,known in the art and described herein, depend upon, for example, thenature of the nucleotide sequence, temperature, and buffer conditions.These conditions also depend on what event is desired, such ashybridization, cleavage, strand extension or transcription.

[0101] Sequence “mutation,” as used herein, refers to any sequencealteration in a sequence of interest in comparison to a referencesequence. A reference sequence can be a wild type sequence or a sequenceto which one wishes to compare a sequence of interest. A sequencemutation includes single nucleotide changes, or alterations of more thanone nucleotide in a sequence, due to mechanisms such as substitution,deletion or insertion. Single nucleotide polymorphism (SNP) is also asequence mutation as used herein.

[0102] “Single stranded conformation polymorphism,” and “SSCP,” as usedherein, generally refer to the specific conformation of a singlestranded nucleic acid as is affected by its specific nucleic acidsequence. Alteration of the sequence of the single strandedpolynucleotide, such as single nucleotide substitution, deletions orinsertions, result in change, or polymorphism, of the conformation ofthe single stranded polynucleotide. The conformation of thepolynucleotide is generally detectable, identifiable and/ordistinguishable using methods known in the art, such as electrophoreticmobility as measured by gel electrophoresis, capillary electrophoresis,and/or susceptibility to endonuclease digestion.

[0103] “Microarray” and “array,” as used interchangeably herein,comprise a surface with an array, preferably ordered array, of putativebinding (e.g., by hybridization) sites for a biochemical sample (target)which often has undetermined characteristics. In a preferred embodiment,a microarray refers to an assembly of distinct polynucleotide oroligonucleotide probes immobilized at defined positions on a substrate.Arrays are formed on substrates fabricated with materials such as paper,glass, ceramic, plastic (e.g., polypropylene, nylon, polystyrene),polyacrylamide, nitrocellulose, silicon or other metals, optical fiberor any other suitable solid or semisolid support, and configured in aplanar (e.g., glass plates, silicon chips) or three-dimensional (e.g.,pins, fibers, beads, particles, microtiter wells, capillaries)configuration. Probes forming the arrays may be attached to thesubstrate by any number of ways including (i) in situ synthesis (e.g.,high-density oligonucleotide arrays) using photolithographic techniques(see, Fodor et al., Science (1991), 251:767-773; Pease et al., Proc.Natl. Acad. Sci. U.S.A. (1994), 91:5022-5026; Lockhart et al., NatureBiotechnology (1996), 14:1675; U.S. Pat. Nos. 5,578,832; 5,556,752; and5,510,270); (ii) spotting/printing at medium to low-density (e.g., cDNAprobes) on glass, nylon or nitrocellulose (Schena et al, Science (1995),270:467-470, DeRisi et al, Nature Genetics (1996), 14:457-460; Shalon etal., Genome Res. (1996), 6:639-645; and Schena et al., Proc. Natl. Acad.Sci. U.S.A. (1995), 93:10539-11286); (iii) by masking (Maskos andSouthern, Nuc. Acids. Res. (1992), 20:1679-1684) and (iv) bydot-blotting on a nylon or nitrocellulose hybridization membrane (see,e.g., Sambrook et al., Eds., 1989, Molecular Cloning: A LaboratoryManual, 2nd ed., Vol. 1-3, Cold Spring Harbor Laboratory (Cold SpringHarbor, N.Y.)). Probes may also be noncovalently immobilized on thesubstrate by hybridization to anchors, by means of magnetic beads, or ina fluid phase such as in microtiter wells or capillaries. The probemolecules are generally nucleic acids such as DNA, RNA, PNA, and cDNAbut may also include proteins, polypeptides, oligosaccharides, cells,tissues and any permutations thereof which can specifically bind thetarget molecules.

[0104] The term “3′” generally refers to a region or position in apolynucleotide or oligonucleotide 3′ (downstream) from another region orposition in the same polynucleotide or oligonucleotide.

[0105] The term “5′” generally refers to a region or position in apolynucleotide or oligonucleotide 5′ (upstream) from another region orposition in the same polynucleotide or oligonucleotide.

[0106] The term “3′-DNA portion,” “3′-DNA region,” “3′-RNA portion,” and“3′-RNA region,” refer to the portion or region of a polynucleotide oroligonucleotide located towards the 3′ end of the polynucleotide oroligonucleotide, and may or may not include the 3′ most nucleotide(s) ormoieties attached to the 3′ most nucleotide of the same polynucleotideor oligonucleotide. The 3′ most nucleotide(s) can be preferably fromabout 1 to about 20, more preferably from about 3 to about 18, even morepreferably from about 5 to about 15 nucleotides.

[0107] The term “5′-DNA portion,” “5′-DNA region,” “5′-RNA portion,” and“5′-RNA region,” refer to the portion or region of a polynucleotide oroligonucleotide located towards the 5′ end of the polynucleotide oroligonucleotide, and may or may not include the 5′ most nucleotide(s) ormoieties attached to the 5′ most nucleotide of the same polynucleotideor oligonucleotide. The 5′ most nucleotide(s) can be preferably fromabout 1 to about 20, more preferably from about 3 to about 18, even morepreferably from about 5 to about 15 nucleotides.

[0108] As is well understood by those skilled in the art, a “tail”sequence of a primer is a sequence not hybridizable to the targetsequence under conditions in which other region(s) or portion(s) of theprimer hybridizes to the target.

Amplifcation Methods of the Invention

[0109] The following are examples of the amplification methods of theinvention. It is understood that various other embodiments may bepracticed, given the general description provided above.

[0110] Methods of generating multiple copies of (amplifying) an RNAsequence complementary to an RNA sequence of interest are provided. Insome aspect, the amplification methods of the invention include atranscription step. In a first embodiment of these methods, linearnucleic acid amplification is achieved based on hybridizing apropromoter polynucleotide to a primer extension product to generate anintermediate product capable of driving transcription, whereby RNAtranscripts comprising sequences complementary to an RNA sequence ofinterest are produced. In a second embodiment of these methods,exponential amplification is achieved by subjecting amplified RNAproducts generated in the process of the first embodiment of this methodand in subsequent amplification to cyclical amplification.

[0111] In embodiments of the amplification methods of the inventionwhich include a transcription step generally provide as follows: if aprimer extension product that is to be transcribed comprises apropromoter sequence, a double stranded promoter region is generallygenerated by hybridizing a polynucleotide comprising a propromoter (alsoreferred to herein as “propromoter polynucleotide”) to the primerextension product. If a primer extension product does not comprise adesired propromoter sequence, the transcription step is generallydependent on the incorporation of an RNA polymerase propromotersequence, by use of a propromoter polynucleotide such as a promotersequence oligonucleotide (PTO). A propromoter polynucleotide such as thePTO can serve as a template for extension of a single stranded primerextension product and formation of a partial duplex comprising a doublestranded promoter at one end. The ability to hybridize a single strandedprimer extension product to the propromoter polynucleotide (such as aPTO) is generally achieved by creating a primer extension product with adefined 3′ end sequence, which is complementary to the 3′ end sequenceof the propromoter polynucleotide.

[0112] In another aspect, the invention provides a method for generatingmultiple copies (amplifying) of a polynucleotide (DNA) sequencecomplementary to an RNA sequence of interest using a first primer and acomposite primer.

[0113] One aspect of the methods of the invention includes the design ofprimers which are able to hybridize to RNA sequences, such as aplurality of RNA sequences, for initiation of primer extension andproduction of amplification substrates and products.

Methods of Amplifying an RNA Sequence of Interest Nucleic Acid SequenceAmplification Using a First Primer and a Propromoter Polynucleotide, anda Propromoter Polynucleotide

[0114] The invention provides methods of generating multiple copies ofthe complementary sequence of an RNA sequence of interest by using aprimer that comprises a sequence that when introduced into theamplification steps result in generation of an intermediatepolynucleotide to which a propromoter polynucleotide can hybridize. Theprimer is designed to comprise a sequence the complement of which ishybridizable by a propromoter polynucleotide. Generally, this sequenceis in the 5′ portion of the primer. The sequence can be a sequence (thecomplement of which is hybridizable by the propromoter polynucleotideused) that is hybridizable to a target RNA, or a sequence (thecomplement of which is hybridizable by the propromoter polynucleotideused) that is not hybridizable to a target RNA. In some embodiments,linear amplification is achieved. In other embodiments wherein amplifiedRNA products are subjected to further rounds of amplification, cyclical,and thus exponential, amplification is achieved.

[0115] A schematic exemplary depiction of one embodiment of the linearmethods of the invention is provided in FIG. 1. A schematic depiction ofone embodiment of the exponential amplification methods of the inventionis provided in FIG. 2. An embodiment of the linear amplification methodof the invention illustrated in FIG. 1 employs two oligonucleotideswhich are combined with the sample as shown in the figure: a) a firstprimer (labeled “4”), which can be composed of two portions, a 3′portion (labeled “A”), and a 5′ portion (labeled “B”); and b) a secondprimer (labeled 5”). The exponential method of the invention asillustrated in FIGS. 2A-B employs three oligonucleotides which arecombined with the sample as shown in the figure: a) a first primer(labeled “4”) which can be composed of two portions, a 3′ portion(labeled “A”); and a 5′ portion (labeled “B”); b) a second primer(labeled “5”); and c) a propromoter polynucleotide, such as a promotertemplate oligonucleotide (PTO), (labeled “6”).

[0116] The 3′ portion of the first primer illustrated in FIGS. 1 and 2Acan be designed in any of a number of ways (in terms of sequence),depending on which type, class, population, and/or species of RNA isdesired to be amplified. In some embodiments, the 3′ portion of thefirst primer, as illustrated in FIGS. 1 and 2A, comprises a sequencecomplementary to the poly-A tail of mRNA, and may further compriseadditional random sequences (generally not complementary to a poly-Asequence) at the 3′ end of the 3′ portion. In other embodiments, the 3′portion of the first primer is a random primer comprising sequenceswhich are hybridizable to a multiplicity of RNA species (which may rangefrom 2 or more to many hundred or thousands or more). Random primers areknown in the art, for example, they have been used extensively in thepreparation of cDNA libraries using PCR-based procedures. As is wellunderstood in the art, a “random primer” can refer to a primer that is amember of a population of primers (a plurality of random primers) whichcollectively are designed to hybridize to a desired and/or a significantnumber of target sequences. A random primer may hybridize at a pluralityof sites on a nucleic acid sequence.

[0117] In other embodiments, the 3′ portion of the first primer cancomprise a sequence complementary or hybridizable to a specific RNA orfamily of RNAs (or portions thereof).

[0118] In some embodiments as illustrated in FIGS. 1 and 2A, the 5′portion of the first primer can be a sequence not hybridizable to thetarget sequence (under conditions in which the 3′ portion hybridizes toRNA target), e.g., a sequence forming a “tail” when the primer ishybridized to a target. This “tail” sequence generally is incorporatedinto the first primer extension product (first strand cDNA), and thecomplement of this tail will be incorporated at the 3′ end of the secondprimer extension product (second strand EDNA). Accordingly, in someembodiments, the first primer is a mixture of first primers whichcomprise the same 5′ DNA portion and a multiplicity of 3′ DNA portionsselected to amplify a multiplicity (which can be small to very large) ofRNA sequences of interest. In other embodiments, the 5′ portion of thefirst primer can be hybridizable to the target RNA.

[0119] The second primer as illustrated in FIGS. 1 and 2A may compriserandom sequences, which are known in the art, and that are complementaryto sequences of a plurality of the first cDNA strands produced. The 3′end of the propromoter polynucleotide is preferably, but notnecessarily, non-extendable. The 3′ portion of the PTO generallycomprises a sequence that is typically identical to the B sequence ofthe first primer 4.

[0120] Primer 2 as illustrated in FIGS. 1 and 2A can be, but is notnecessarily, composed of DNA and can comprise two sections(interchangeably called “portions” or “regions”). The 3′ portion ofprimer 2 can be selected for random priming of many, most and/or allpossible mRNA sequences in a biological sample. Random primers are knownin the art, for example, they have been used extensively in thepreparation of cDNA libraries using PCR-based procedures. In someembodiments, the hybridizable sequence of the second primer is designedbased on a known sequence of the desired binding site on a first strandcDNA. In other embodiments, the second primer comprises a strand switcholigonucleotide, described in U.S. Pat. Nos. 5,962,271 and 5,962,272,which is hybridizable to the Cap sequences present on mRNA and causesthe reverse transcriptase to switch from the mRNA template to the switcholigonucleotide, permitting generation of a second strand cDNA primed bythe “switch oligonucleotide”. Alternatively, a homopolymeric tail isadded to the 3′terminus of the first primer extension product, and thesecond primer comprises the complement of the homopolymeric tail.

[0121] In some embodiments, the second primer comprises DNA. In otherembodiments, the second primer consists of DNA. In other embodiments, asdescribed herein, the second primer is a fragment of the target RNA,with the fragment being generated by cleavage of the RNA target.

[0122] The 5′ portion of primer 2 can be a sequence which is notcomplementary and not substantially hybridizable to a specific targetsequence, i.e., it would not hybridize (under conditions in which the 3′portion hybridizes to RNA target) and would constitute a tail. The“tail” sequence would generally be incorporated into the second primerextension product.

[0123] A promoter template oligonucleotide, 3 (PTO), can be designed asfollows: the 3′ end of the propromoter polynucleotide is preferably, butnot necessarily, non-extendable. The 3′ portion of the PTO generallycomprises a sequence that is typically identical to the B sequence ofthe first primer 4. The 5′-most portion of the PTO is a promotersequence for a DNA-dependent RNA polymerase, which, as described above,is used in certain embodiments of the amplification methods of theinvention. Generally, the sequence between these two sections isdesigned for optimal transcription by the polymerase of choice. Criteriafor selection and design of this sequence are known in the art.

[0124] As illustrated in FIG. 1, one embodiment of the process of thelinear amplification methods of the invention is as follows:

[0125] A) Formation of Double Stranded cDNA Substrate from Target RNA

[0126] 1. Primer 4 binds to a target RNA by hybridization of its 3′ endto the target to form complex I. The most commonly used initiation sitefor binding of the first primer to generate first strand cDNA is the 3′end poly-A sequence of mRNA and the immediate adjacent nucleotides.Binding of the primer to these immediate adjacent nucleotides can beachieved by including partially random sequences (other than sequencecomplementary to poly-A sequence of a target mRNA) at the 3′ end of theprimer. Criteria for primer designs for this purpose are known in theart, for example in the selection of primer sequences in the generationof libraries (including cDNA libraries). In cases where it is desired toproduce libraries of sequences of a pre-defined family of mRNA, such asfor example preparation of libraries from an immunoglobulin chain, the Asequence of the first primer could comprise a sequence which is wellpreserved in all members of the family of RNA from the immunoglobulinchain.

[0127] 2. Primer extension along the RNA strand of complex I is carriedout by an RNA-dependent DNA polymerase such as reverse transcriptase(labeled “RT”), to form an RNA/DNA hybrid duplex. RNase H degrades theRNA strand of the hybrid duplex to produce a first cDNA strand (labeled“I”). The RNase H activity may be supplied by the RNA-dependent DNApolymerase (such as reverse transcriptase) or may be provided in aseparate enzyme. Reverse transcriptases useful for this method may ormay not have RNase H activity.

[0128] 3. A second primer (labeled “5”) binds to II at a complementarysite to form complex III. In the case where a plurality of RNA speciesare being amplified simultaneously, the second primers could be randomprimers that bind to II at random complementary sites of a plurality ofcDNA species. When it is desired to generate a library of a known mRNAfamily, such as for example a library of a specific immunoglobulinchain, primer 5 may comprise a sequence which is complementary to a wellconserved sequence in this family.

[0129] 4. Primer extension along the first cDNA strand is carried out bya DNA-dependent DNA polymerase to generate a double stranded cDNA(labeled “IV”) comprising at one end a duplex of sequence B (of thefirst primer) and its complement. The cDNAs are substrates foramplification according to the methods of the invention.

[0130] 5. The double stranded products IV are denatured (for example, byheat) to separate the two DNA strands. Various methods for strandseparation could be employed for carrying out the methods of theinvention. Strand V, which is of the same sense as the input (target)RNA, is a substrate for amplification, and comprises at its 5′ end thesequence of the second primer (primer 5), and at its 3′ end the sequencecomplementary to sequence B of the first primer (primer 4).

[0131] B) Linear Isothermal Amplification

[0132] 1. A propromoter oligonucleotide (such as a PTO) binds to the 3′end of strand V by hybridization of its 3′ end sequence B to itscomplementary sequence at the 3′ end of V, to form complex VI.

[0133] 2. A DNA-dependent DNA polymerase extends the 3′ end of the sensecDNA strand (strand V) in complex VI to form the partial duplex VII,comprising a double stranded RNA polymerase promoter at one end.

[0134] 3. An RNA polymerase binds to the double stranded promoter andtranscribes the cDNA strand of complex VII to produce multiple copies ofsingle stranded antisense RNA products, VIII. These antisense RNAproducts can also serve as substrates for exponential amplification.

[0135] One embodiment of the exponential amplification methods of theinvention is illustrated in FIGS. 2A-B. In this embodiment, subsequentto the generation of antisense RNA products in the linear amplificationsteps, the following steps are performed.

[0136] C) Exponential Isothermal Amplification

[0137] 1. Primer 5 binds to the 3′ end of the RNA products VIII to formcomplex IX.

[0138] 2. Primer extension along the RNA strand is carried out by anRNA-dependent DNA polymerase to form an RNA/DNA hybrid duplex, X.

[0139] 3. RNase H degrades the RNA strand of complex X to produce singlestranded DNA copies of the RNA products. A propromoter polynucleotidesuch as a PTO hybridizes to the 3′ end of the single stranded DNA toform complex XI.

[0140] 4. The 3′ end of the DNA strand in complex XI is extended alongthe propromoter polynucleotide (PTO) to form a partial duplex XII,comprising a double stranded promoter sequence at one end. This productis the same as VII, and is a substrate for RNA polymerase for thegeneration of multiple copies of the RNA products, as in step b(3)above, thus producing a cyclical process for the exponentialamplification of a target RNA.

Nucleic Acid Sequence Amplification Using a First Primer, a CompositePrimer, Denaturation and Strand Displacement

[0141] The invention provides methods of amplifying an RNA sequence ofinterest by using a first primer and a composite primer, denaturation,and strand displacement. Amplification can be achieved isothermally,though every step is not necessarily conducted at the same temperature.Amplified products are single stranded DNA comprising sequencescomplementary to the RNA sequence of interest in the target RNA.

[0142] A schematic description of one embodiment of the compositeprimer, second primer and strand displacement-based methods of theinvention is given in FIGS. 3A and B. The methods involve the followingsteps: (a) formation of a second strand cDNA which is the same sense asthe input RNA (as described herein, and one embodiment of which isillustrated in FIG. 1); and (b) linear amplification of the complementof a second strand cDNA strand by primer extension from a compositeprimer along the second strand cDNA and strand displacement. See Kurn,U.S. Pat. No. 6,251,639 B1.

[0143] The embodiment illustrated in FIGS. 3A and 3B employs threeoligonucleotides: a first primer which can be composed of two portions,a 3′ portion (labeled “A”); and a 5′ portion (labeled “B”), (labeled 1),used for formation of first strand cDNA; a second primer, used for theformation of the second strand cDNA (which may be an exogenously addedsecond primer or one or more fragments of target RNA that remainshybridized to the first strand cDNA following RNase H treatment); and acomposite primer used for linear isothermal amplification. The compositeprimer comprises a 5′ RNA portion and a DNA portion. The first andsecond primers used in this aspect of the invention may comprise anyfirst and second primer described herein (including a second primercomprising one or more fragments .

[0144] The composite primer illustrated in FIG. 3A and B comprises asequence capable of hybridizing to the second strand cDNA, and mostoften comprises a sequence hybridizable to the defined 3′-portion of thesecond strand cDNA (that is the complement of the first primersequence). The composite primer may additionally comprise sequences nothybridizable to the second strand cDNA under conditions which thecomposite primer hybridizes, such that a tail is formed.

[0145] As illustrated in FIGS. 3A and B, the composite primer comprisesa DNA portion at its 3′ end, and an RNA portion at its 5′ end. Asdiscussed herein, it is also possible to employ a composite primer inwhich the 3′ DNA portion is followed, in the direction of its 5′, by anRNA portion, which is followed by a portion which is DNA. The length ofeach of these sections is generally determined for maximum efficiency ofthe amplification. Only the two-portion (i.e., 3′-DNA-RNA-5′) compositeprimer is shown in FIGS. 3A and B.

[0146] As illustrated in FIGS. 3A and B, in one embodiment, the processof the amplification methods of the invention resulting in generation ofDNA products comprising sequences complementary to an RNA sequence(s) ofinterest based on RNA is as follows:

[0147] A) Formation of a Single Stranded cDNA Substrate forAmplification Using a Composite Primer

[0148] 1. A primer binds to a target RNA by hybridization of its 3′ endto the target.

[0149] 2. Primer extension along the RNA strand is carried out by anRNA-dependent DNA polymerase such as reverse transcriptase, to form anRNA/DNA hybrid duplex. RNase H degrades the RNA strand of the hybridduplex to produce a first cDNA strand (labeled “I”). The RNase Hactivity may be supplied by the RNA-dependent DNA polymerase (such asreverse transcriptase) or may be provided in a separate enzyme. Reversetranscriptases useful for this method may or may not have RNase Hactivity.

[0150] 3. Primer extension along the first cDNA strand is carried out bya DNA-dependent DNA polymerase (not illustrated in FIG. 3A) to generatea double stranded cDNA, forming a complex of first and second strandcDNAs (wherein the second strand cDNA comprises a 3′ end portion that isthe complement of the first primer sequence), as shown in FIG. 3A.

[0151] 4. The complex of first and second strand cDNAs is then denaturedto form single stranded first strand cDNA and second strand cDNA, asshown in FIG. 3B. The single stranded second strand cDNA is thesubstrate for isothermal amplification using a composite primer andstrand displacement as follows.

[0152] B) Generation of Bouble Stranded cDNA Comprising an RNA/DNAHeteroduplex

[0153] 1. A composite primer comprising a 5′-RNA portion and a DNAportion hybridizes to the 3′-portion of the second strand cDNA,generally to the 3′-portion of the second strand cDNA, and is extendedalong the second strand cDNA by a DNA polymerase to form a doublestranded complex comprising an RNA/DNA hybrid portion at one end of thecomplex.

[0154] C) Isothermal Linear Amplification

[0155] 1. An agent, such as an enzyme, which cleaves RNA from an RNA/DNAhybrid (such as RNase H) cleaves RNA sequence from the hybrid, leaving asequence on the second strand cDNA available for binding by anothercomposite primer.

[0156] 2. Composite primer binds by hybridization of the RNA portion tothe single stranded DNA end on the second strand cDNA, which iscomplementary to it. The 3′ DNA sequence of primer is not hybridized.

[0157] 3. The 3′ end of bound composite primer, and the 5′ end of theDNA strand immediately upstream are the same, and would compete forhybridization to the opposite strand. Without wishing to be bound bytheory, the high affinity of the DNA polymerase to hybridized 3′ end ofa primer would be expected to push the equilibrium of the two competingstructures towards hybridization of the 3′ end of the new primer anddisplacement of the 5′ end of the previous primer extension product.Primer extension along the second strand (sense) cDNA strand results indisplacement of the previous second strand cDNA, and formation of adouble stranded cDNA having, at one end, an RNA/DNA hybrid composed ofsequence B and its complement

[0158] 4. The RNA segment of the hybrid is degraded by agent, such asRNase H, which results in the formation of a single stranded 3′ end towhich a new composite primer can be bound by its 5′ portion.

[0159] 5. The process of hybridization of the 3′ end sequence of thebound composite primer, by displacement of the 5′-most end of theprevious primer extension product in the duplex, primer extension anddisplacement of the previous product continues, and results in theaccumulation of multiple copies of anti-sense single stranded DNAproducts.

[0160] In some embodiments, subsequent to the generation of antisenseRNA products in the linear amplification steps, the following steps areperformed:

[0161] D) Exponential Isothermal Amplification

[0162] 1. A propromoter oligonucleotide (such as a PTO) binds to the 3′end of anti-sense single stranded DNA products.

[0163] 2. Primer extension along the RNA strand is carried out by anRNA-dependent DNA polymerase to form an RNA/DNA hybrid duplex. Thisproduct is the same is a substrate for RNA polymerase for the generationof multiple copies of sense RNA products. As described herein, extensionof the propromoter polynucleotide may or may not be required to effectcreation of a propromoter for transcription.

Components and Reaction Conditions Used in the Methods of the InventionTemplate Nucleic Acid

[0164] The RNA target to be amplified includes RNAs from any source inpurified or unpurified form, which can be RNA such as total RNA, tRNA,mRNA, rRNA, mitochondrial RNA, chloroplast RNA, DNA-RNA hybrids, ormixtures thereof, from any source and/or species, including human,animals, plants, and microorganisms such as bacteria, yeasts, viruses,viroids, molds, fungi, plants, and fragments thereof. RNAs can beobtained and purified using standard techniques in the art.Amplification of a DNA target would require initial transcription of theDNA target into RNA form, which can be achieved by techniques (such asexpression systems) known in the art. Amplification of a DNA-RNA hybridwould require denaturation of the hybrid to obtain a ssRNA, ordenaturation followed by transcription of the DNA strand to obtain anRNA. The target RNA can be only a minor fraction of a complex mixturesuch as a biological sample and can be obtained from various biologicalmaterial by procedures well known in the art.

[0165] The target RNA can be known or unknown and may contain more thanone desired specific nucleic acid sequence of interest, each of whichmay be the same or different from each other. Therefore, theamplification process is useful not only for producing large amounts ofone specific nucleic acid sequence, but also for amplifyingsimultaneously more than one different specific nucleic acid sequencelocated on the same or different nucleic acid molecules.

[0166] The initial step of the amplification of a target RNA sequence isrendering the target single stranded. Denaturation may be carried out toremove secondary structure present in a RNA target molecule. Thedenaturation step may be thermal denaturation or any other method knownin the art.

First Primer

[0167] The first primer is a primer that comprises a sequence (which mayor may not be the whole of the primer) that is hybridizable (under agiven set of conditions) to the target RNA. This sequence can be basedon a specific sequence of the target, or a random sequence (in someembodiments, the first primer is a random primer). It can also be basedon a general, more universal sequence known to be present in an RNAspecies of interest, such as the poly-A sequence found in mRNA. In someembodiments, the primer may comprise a sequence complementary to apoly-A sequence, and may further comprise a random sequence 3′ to saidsequence complementary to a poly-A sequence. In some embodiments, theprimer comprises a sequence, preferably at the 5′ end, that is nothybridizable (under a given set of conditions) to a target RNA. Inaddition, the sequence that is capable of hybridizing to the target RNAmay comprise a sequence complementary to the poly-A tail of mRNA, andmay further comprise additional random sequences (generally notcomplementary to a poly-A sequence) at the 3′ end of the 3′ portion.

[0168] Random primers are well known in the art, and include at leastthe following: primers hybridizable to two or more sequences in asample; and primers comprising poly-T sequences that are hybridizable toa multiplicity of RNAs in a sample (such as all mRNA). For convenience,a single random composite primer is discussed above. However, it isunderstood that the term “random primer” can refer to a primer that is amember of a population of primers which are collectively designed to adesired and/or significant population of target sequences.

[0169] It is also understood that the amplification of a plurality ofmRNA species in a single reaction mixture may, but not necessarily,employ a multiplicity, or a large multiplicity of primers. Thus, theinvention contemplates the use of a multiplicity of different compositeprimers (random or non-random) when amplifying a plurality of mRNAspecies in a single reaction mixture.

[0170] To achieve hybridization to a target nucleic acid (which, as iswell known and understood in the art, depends on other factors such as,for example, ionic strength and temperature), the sequence of the primerthat is hybridizable to the target RNA is preferably of at least about60%, more preferably at least about 75%, even more preferably at leastabout 90%, and most preferably at least about 95% complementarily to thetarget nucleic acid.

[0171] In some embodiments, the first primer comprises a 5′ sequence(which generally includes the 5′ most nucleotide) the complement ofwhich is hybridizable by a propromoter polynucleotide. This sequenceenables the creation of a defined end sequence for the 5′ end of thefirst primer extension product (and thus, subsequently the 3′ end of thesecond/third primer extension product). Having a defined end sequence isparticularly advantageous with respect to hybridization of a propromoterpolynucleotide to the 3′ end of the second and third primer extensionproducts in subsequent steps. Thus, in these embodiments, the firstprimer comprises a sequence that when introduced into the amplificationsteps of the methods of the invention results in generation of anintermediate polynucleotide to which a propromoter polynucleotide canhybridize. In some of these embodiments, the 5′ sequence the complementof which is hybridizable by a propromoter polynucleotide is hybridizable(under a given set of conditions) to a target RNA when the primer ishybridized to the target RNA. In other embodiments, the 5′ sequence thecomplement of which is hybridizable by a propomoter polynucleotide isnot hybridizable (under a given set of conditions) to a target RNA whenthe primer is hybridized to the target RNA (thus constituting a tailwhen the 3′ sequence of the primer is hybridized to the target).

[0172] In one embodiment, the first primer comprises DNA. In anotherembodiment, the first primer comprises RNA. In yet another embodiment,the first primer comprises DNA and RNA.

Second Primer

[0173] The second primer in the methods of the invention comprises asequence (which may or may not be the whole of the primer) that ishybridizable (under a given set of conditions) to a first primerextension product at a site on the first primer extension product suchthat the second primer extension product would include the RNA sequenceof interest. In some embodiments, the hybridizable sequence of thesecond primer is designed based on a known sequence of the desiredbinding site on a first primer extension product. In other embodiments,the hybridizable sequence is based on random sequences known in the artto be suitable for random priming of a plurality of RNA species. In someembodiments, a second primer comprises a sequence, preferably at the 5′end, that is not hybridizable (under a given set of conditions) to afirst primer extension product. In other embodiments, the second primercomprises a strand switch oligonucleotide, described in U.S. Pat. Nos.5,962,271 and 5,962,272, which is hybridizable to the Cap sequencespresent on mRNA and causes the reverse transcriptase to switch from themRNA template to the switch oligonucleotide, permitting generation of asecond strand cDNA primed by the “switch oligonucleotide”.Alternatively, a homopolymeric tail is added to the 3′ terminus of thefirst primer extension product, and the second primer comprises thecomplement of the homopolymeric tail.

[0174] In one embodiment, the second primer comprises DNA. In anotherembodiment, the second primer consists of DNA. In another embodiment,the second primer comprises RNA. In yet another embodiment, the secondprimer comprises DNA and RNA.

[0175] In some embodiments, the second primer is provided by selfpriming (for example, by a hairpin loop) at the 3′ end of the firstprimer extension product. In these embodiments, a sequence at the 3′ endof the first primer extension product hybridizes to another sequence inthe first primer extension product, for example as described in U.S.Pat. No. 6,132,997. In these embodiments, said sequence at the 3′ of thefirst primer extension product is generally cleaved (for example, withan S1 nuclease) following its hybridization to the first primerextension product and/or its extension along the first primer extensionproduct. See U.S. Pat. No. 6,132,997.

[0176] In some embodiments, the second primer is provided by a targetRNA fragment. Such a target RNA fragment can be generated as a result ofincomplete degradation of a target RNA in a complex of target RNA andfirst primer extension product by an enzyme that cleaves RNA in anRNA/DNA hybrid, such that one or more RNA fragments remain bound to thefirst primer extension product.

[0177] To achieve hybridization to a first primer extension product(which, as is well known and understood in the art, depends on otherfactors such as, for example, ionic strength and temperature), thesequence of the second primer that is hybridizable to the first primerextension product is preferably of at least about 60%, more preferablyat least about 75%, even more preferably at least about 90%, and mostpreferably at least about 95% complementarity to the first primerextension product.

Third Primer

[0178] The third primer in the methods of the invention comprises asequence (which may or may not be the whole of the primer) that ishybridizable (under a given set of conditions) to the RNA transcriptgenerated from the second primer extension product (as the template) ata site on the RNA transcript such that the third primer extensionproduct would include the RNA sequence of interest, if present. In someembodiments, the hybridizable sequence of the third primer is designedbased on a known sequence of the desired binding site on an RNAtranscript. In other embodiments, the hybridizable sequence is based onrandom sequences known in the art to be suitable for random priming of aplurality of RNA species. In some embodiments, the third primercomprises a sequence, preferably at the 5′ end, that is not hybridizable(under a given set of conditions) to an RNA transcript.

[0179] To achieve hybridization to an RNA transcript (which, as is wellknown and understood in the art, depends on other factors such as, forexample, ionic strength and temperature), the sequence of the thirdprimer that is hybridizable to the RNA transcript is preferably of atleast about 60%, more preferably at least about 75%, even morepreferably at least about 90%, and most preferably at least about 95%complementarity to the RNA transcript.

[0180] In one embodiment, the third primer comprises DNA. In anotherembodiment, the third primer comprises RNA. In yet another embodiment,the third primer comprises DNA and RNA.

[0181] In some embodiments, the third primer is provided by self priming(for example, by a hairpin loop) at the 3′ end of an RNA transcript. Inthese embodiments, a sequence at the 3′ end of the RNA transcripthybridizes to another sequence in the RNA transcript itself. In theseembodiments, said sequence at the 3′ of the RNA transcript is generallycleaved following its hybridization to the RNA transcript and/or itsextension along the RNA transcript.

Composite Primer

[0182] In some embodiments, the methods of the invention employ acomposite primer that is composed of RNA and DNA portions. The compositeprimer is designed such that subsequent displacement of the primerextension product by binding of a new (additional) composite primer andthe extension of the new primer by the polymerase can be achieved. Inaddition, cleavage of the RNA portion of the primer extension productleads to generation of amplification product which is not a substratefor amplification by the composite primer.

[0183] The composite primer illustrated in FIG. 3 comprises sequencescapable of hybridizing to the second strand cDNA, and most oftencomprises sequences hybridizable to the defined 3′-portion of the secondstrand cDNA (that is the complement of the first primer sequence). Thecomposite primer may comprise all or a portion of the sequence of thefirst primer. The composite primer may additionally comprise sequencesnot hybridizable to the second strand cDNA such that a tail is formed.

[0184] It is also understood that the amplification of a plurality ofmRNA species in a single reaction mixture may, but not necessarily,employ a multiplicity, or a large multiplicity of primers. Thus, theinvention contemplates the use of a multiplicity of different compositeprimers (random or non-random) when amplifying a plurality of mRNAspecies in a single reaction mixture.

[0185] A composite primer comprises at least one RNA portion that iscapable of (a) binding (hybridizing) to a sequence on the second strandcDNA product independent of hybridization of the DNA portion(s) to asequence on the same extension product; and (b) being cleaved with aribonuclease when hybridized to the second strand cDNA. The compositeprimers bind to the second strand cDNA to form a partial heteroduplex inwhich only the RNA portion of the primer is cleaved upon contact with aribonuclease such as RNase H, while the second strand cDNA remainsintact, thus enabling annealing of another composite primer.

[0186] The composite primers also comprise a 3′ DNA portion that iscapable of hybridization to a sequence on the second strand cDNA suchthat its hybridization to the cDNA is favored over that of the nucleicacid strand that is displaced from the second strand cDNA by the DNApolymerase. Such primers can be rationally designed based on well knownfactors that influence nucleic acid binding affinity, such as sequencelength and/or identity, as well as hybridization conditions. In apreferred embodiment, hybridization of the 3′ DNA portion of thecomposite primer to its complementary sequence in the second strand cDNAis favored over the hybridization of the homologous sequence in the 5′end of the displaced strand to the second strand cDNA.

[0187] Generation of primers suitable for extension by polymerization iswell known in the art, such as described in PCT Pub. No. WO 99/42618(and references cited therein). The composite primer comprises acombination of RNA and DNA (see definition above), with the 3′-endnucleotide being a nucleotide suitable for nucleic acid extension. The3′-end nucleotide can be any nucleotide or analog that when present in aprimer, is extendable by a DNA polymerase. Generally, the 3′-endnucleotide has a 3′-OH. Suitable primers include those that comprise atleast one portion of RNA and at least one portion of DNA. For example,composite primers can comprise a 5′-RNA portion and a 3′-DNA portion (inwhich the RNA portion is adjacent to the 3′-DNA portion); or 5′- and3′-DNA portions with an intervening RNA portion. Accordingly, in oneembodiment, the composite primer comprises a 5′ RNA portion and a 3′-DNAportion, preferably wherein the RNA portion is adjacent to the 3′-DNAportion. In another embodiment, the composite primer comprises 5′ and3′-DNA portions with at least one intervening RNA portion (i.e., an RNAportion between the two DNA portions). In yet another embodiment, thecomposite primer of the invention comprises a 3′-DNA portion and atleast one intervening RNA portion (i.e., an RNA portion between DNAportions).

[0188] The length of an RNA portion in a composite primer comprising a3′-DNA portion and an RNA portion can be preferably from about 1 toabout 50, more preferably from about 3 to about 20, even more preferablyfrom about 4 to about 15, and most preferably from about 5 to about 10nucleotides. In some embodiments of a composite primer comprising a3′-DNA portion and an RNA portion, an RNA portion can be at least aboutany of 1, 3, 4, 5 nucleotides, with an upper limit of about any of 10,15, 20, 25, 3, 50 nucleotides.

[0189] The length of the 5′-RNA portion in a composite primer comprisinga 5′-RNA portion and a 3′-DNA portion can be preferably from about 3 toabout 50 nucleotides, more preferably from about 5 to about 20nucleotides, even more preferably from about 7 to about 18 nucleotides,preferably from about 8 to about 17 nucleotides, and most preferablyfrom about 10 to about 15 nucleotides. In other embodiments of acomposite primer comprising a 5′-RNA portion and a 3′-DNA portion, the5′-RNA portion can be at least about any of 3, 5, 7, 8, 10 nucleotides,with an upper limit of about any of 15, 17, 18, 20, 50 nucleotides.

[0190] In embodiments of a composite primer comprising a 5′-RNA portionand a 3′-DNA portion further comprising non-5′-RNA portion(s), anon-5′-RNA portion can be preferably from about 1 to about 7nucleotides, more preferably from about 2 to about 6 nucleotides, andmost preferably from about 3 to about 5 nucleotides. In certainembodiments of a composite primer comprising a 5′-RNA portion and a3′-DNA portion further comprising non-5′-RNA portion(s), a non-5′-RNAportion can be at least about any of 1, 2, 3, 5, with an upper limit ofabout any of 5, 6, 7, 10 nucleotides.

[0191] In embodiments of a composite primer comprising a 5′-RNA portionand a 3′-DNA portion, in which the 5′-RNA portion is adjacent to the3′-DNA portion, the length of the 5′-RNA portion can be preferably fromabout 3 to about 50 nucleotides, more preferably from about 5 to about20 nucleotides, even more preferably from about 7 to about 18nucleotides, preferably from about 8 to about 17 nucleotides, and mostpreferably from about 10 to about 15 nucleotides. In certain embodimentsof a composite primer comprising a 5′-RNA portion and a 3′-DNA portion,in which the 5′-RNA portion is adjacent to the 3′-DNA portion, the5′-RNA portion can be at least about any of 3, 5, 7, 8, 10 nucleotides,with an upper limit of about any of 15, 17, 18, 20, 50 nucleotides.

[0192] The length of an intervening RNA portion in a composite primercomprising 5′- and 3′-DNA portions with at least one intervening RNAportion can be preferably from about 1 to about 7 nucleotides, morepreferably from about 2 to about 6 nucleotides, and most preferably fromabout 3 to about 5 nucleotides. In some embodiments of a compositeprimer comprising 5′- and 3′-DNA portions with at least one interveningRNA portion, an intervening RNA portion can be at least about any of 1,2, 3, 5 nucleotides, with an upper limit of about any of 5, 6, 7, 10nucleotides. The length of an intervening RNA portion in a compositeprimer comprising a 3′-DNA portion and at least one intervening RNAportion can be preferably from about 1 to about 7 nucleotides, morepreferably from about 2 to about 6 nucleotides, and most preferably fromabout 3 to about 5 nucleotides. In some embodiments of a compositeprimer comprising a 3′-DNA portion and at least one intervening RNAportion, an intervening RNA portion can be at least about any of 1, 2,3, 5 nucleotides, with an upper limit of about any of 5, 6, 7, 10nucleotides. In a composite primer comprising a 3′-DNA portion and atleast one intervening RNA portion, further comprising a 5′-RNA portion,the 5′-RNA portion can be preferably from about 3 to about 25nucleotides, more preferably from about 5 to about 20 nucleotides, evenmore preferably from about 7 to about 18 nucleotides, preferably fromabout 8 to about 17 nucleotides, and most preferably from about 10 toabout 15 nucleotides. In some embodiments of a composite primercomprising a 3′-DNA portion and at least one intervening RNA portion,further comprising a 5′-RNA portion, the 5′-RNA portion can be at leastabout any of 3, 5, 7, 8, 10 nucleotides, with an upper limit of aboutany of 15, 17, 18, 20 nucleotides.

[0193] The length of the 3′-DNA portion in a composite primer comprisinga 3′-DNA portion and an RNA portion can be preferably from about 1 toabout 20, more preferably from about 3 to about 18, even more preferablyfrom about 5 to about 15, and most preferably from about 7 to about 12nucleotides. In some embodiments of a composite primer comprising a3′-DNA portion and an RNA portion, the 3′-DNA portion can be at leastabout any of 1, 3, 5, 7, 10 nucleotides, with an upper limit of aboutany of 10, 12, 15, 18, 20, 22 nucleotides.

[0194] The length of the 3′-DNA portion in a composite primer comprisinga 5′-RNA portion and a 3′-DNA portion can be preferably from about 1 toabout 20 nucleotides, more preferably from about 3 to about 18nucleotides, even more preferably from about 5 to about 15 nucleotides,and most preferably from about 7 to about 12 nucleotides. In someembodiments of a composite primer comprising a 5′-RNA portion and a3′-DNA portion, the 3′ DNA portion can be at least about any of 1, 3, 5,7, 10 nucleotides, with an upper limit of about any of 10, 12, 15, 18,20, 22 nucleotides.

[0195] In embodiments of a composite primer comprising a 5′-RNA portionand a 3′-DNA portion, further comprising non-3′-DNA portion(s), anon-3′-DNA portion can be preferably from about 1 to about 10nucleotides, more preferably from about 2 to about 8 nucleotides, andmost preferably from about 3 to about 6 nucleotides. In some embodimentsof a composite primer comprising a 5′-RNA portion and a 3′-DNA portion,further comprising non-3′-DNA portion(s), a non-3′-DNA portion can be atleast about any of 1, 2, 3, 5 nucleotides, with an upper limit of aboutany of 6, 8, 10, 12 nucleotides.

[0196] In embodiments of a composite primer comprising a 5′-RNA portionand a 3′-DNA portion in which the 5′-RNA portion is adjacent to the3′-DNA portion, the length of the 3′-DNA portion can be preferably fromabout 1 to about 20 nucleotides, more preferably from about 3 to about18 nucleotides, even more preferably from about 5 to about 15nucleotides, and most preferably from about 7 to about 12 nucleotides.In certain embodiments of the primer comprising a 5′-RNA portion and a3′-DNA portion in which the 5′-RNA portion is adjacent to the 3′-DNAportion, the 3′-DNA portion can be at least about any of 1, 3, 5, 7, 10nucleotides, with an upper limit of about any of 10, 12, 15, 18, 20, 22nucleotides.

[0197] The length of a non-3′-DNA portion in a composite primercomprising 5′- and 3′-DNA portions with at least one intervening RNAportion can be preferably from about 1 to about 10 nucleotides, morepreferably from about 2 to about 8 nucleotides, and most preferably fromabout 3 to about 6 nucleotides. In some embodiments of a primercomprising 5′- and 3′-DNA portions with at least one intervening RNAportion, a non-3′-DNA portion can be at least about any of 1, 2, 3, 5nucleotides, with an upper limit of about any of 6, 8, 10, 12nucleotides.

[0198] The length of the 3′-DNA portion in a composite primer comprising5′- and 3′-DNA portions with at least one intervening RNA portion can bepreferably from about 1 to about 20 nucleotides, more preferably fromabout 3 to about 18 nucleotides, even more preferably from about 5 toabout 15 nucleotides, and most preferably from about 7 to about 12nucleotides. In some embodiments of a composite primer comprising 5′-and 3′-DNA portions with at least one intervening RNA portion, the3′-DNA portion can be at least about any of 1, 3, 5, 7, 10 nucleotides,with an upper limit of about any of 10, 12, 15, 18, 20, 22 nucleotides.

[0199] The length of a non-3′-DNA portion (i.e., any DNA portion otherthan the 3′-DNA portion) in a composite primer comprising a 3′-DNAportion and at least one intervening RNA portion can be preferably fromabout 1 to about 10 nucleotides, more preferably from about 2 to about 8nucleotides, and most preferably from about 3 to about 6 nucleotides. Insome embodiments of a composite primer comprising a 3′-DNA portion andat least one intervening RNA portion, a non-3′-DNA portion can be atleast about any of 1, 3, 5, 7, 10 nucleotides, with an upper limit ofabout any of 6, 8, 10, 12 nucleotides. The length of the 3′-DNA portionin a composite primer comprising a 3′-DNA portion and at least oneintervening RNA portion can be preferably from about 1 to about 20nucleotides, more preferably from about 3 to about 18 nucleotides, evenmore preferably from about 5 to about 15 nucleotides, and mostpreferably from about 7 to about 12 nucleotides. In some embodiments ofa composite primer comprising a 3′-DNA portion and at least oneintervening RNA portion, the 3′-DNA portion can be at least about any of1, 3, 5, 7, 10 nucleotides, with an upper limit of about any of 10, 12,15, 18, 20, 22 nucleotides. It is understood that the lengths for thevarious portions can be greater or less, as appropriate under thereaction conditions of the methods of this invention.

[0200] In some embodiments, the 5′-DNA portion of a composite primerincludes the 5′-most nucleotide of the primer. In some embodiments, the5′-RNA portion of a composite primer includes the 5′ most nucleotide ofthe primer. In other embodiments, the 3′-DNA portion of a compositeprimer includes the 3′ most nucleotide of the primer. In otherembodiments, the 3′-DNA portion is adjacent to the 5′-RNA portion andincludes the 3′ most nucleotide of the primer (and the 5′-RNA portionincludes the 5′ most nucleotide of the primer).

[0201] The total length of the composite primer can be preferably fromabout 10 to about 50 nucleotides, more preferably from about 15 to about30 nucleotides, and most preferably from about 20 to about 25nucleotides. In some embodiments, the length can be at least about anyof 10, 15, 20, 25 nucleotides, with an upper limit of about any of 25,30, 50, 60 nucleotides. It is understood that the length can be greateror less, as appropriate under the reaction conditions of the methods ofthis invention.

[0202] To achieve hybridization to a target nucleic acid (which, as iswell known and understood in the art, depends on other factors such as,for example, ionic strength and temperature), the portion of the primerthat is hybridizable to the target RNA is preferably of at least about60%, more preferably at least about 75%, even more preferably at leastabout 90%, and most preferably at least about 95% complementarity to thetarget nucleic acid.

Polynucleotide Comprising a Propromoter and a Region Which Hybridizes toa Primer Extension Product

[0203] The methods of the invention employ a propromoter polynucleotidecomprising a propromoter and a region which hybridizes to a primerextension product. In some embodiments, the propromoter polynucleotideis provided as a PTO, as described in greater detail below.

Propromoter Template Oligonucleotide

[0204] In some embodiments, the methods employ a promoter sequence fortranscription which is provided by a propromoter templateoligonucleotide (PTO).

[0205] A PTO for use in the methods and compositions of the invention isa single-stranded polynucleotide, generally DNA, comprising apropromoter sequence that is designed for formation of a double strandedpromoter of an RNA polymerase, and a portion capable of hybridizing tothe 3′ end of a primer extension product. In a preferred embodiment, thepropromoter sequence is located in the 5′ portion of the oligonucleotideand the hybridizing sequence is located in the 3′ portion of theoligonucleotide. In one embodiment, and most typically, the promoter andhybridizing sequences are different sequences. In another embodiment,the promoter and hybridizing sequences overlap in sequence identity. Inyet another embodiment, the promoter and hybridizing sequences are thesame sequence, and thus are in the same location on the PTO. In theembodiments wherein hybridization of the PTO to the primer extensionproduct results in a duplex comprising an overhang (the 5′ end of thePTO that does not hybridize to the primer extension product, typicallycomprising all or part of the propromoter sequence), DNA polymerasefills in the overhang to create a double stranded promoter capable ofeffecting transcription by a suitable RNA polymerase.

[0206] Promoter sequences that allow transcription of a template DNA areknown in the art and have been discussed above. Preferably, the promotersequence is selected to provide optimal transcriptional activity of theparticular RNA polymerase used. Criteria for such selection, i.e., aparticular promoter sequence particularly favored by a particular RNApolymerase, is also known in the art. For example, the sequences of thepromoters for transcription by T 7 DNA dependent RNA polymerase and SP 6are known in the art. The promoter sequence can be from a prokaryotic oreukaryotic source.

[0207] In some embodiments, the PTO comprises an intervening sequencebetween a propromoter sequence and a portion capable of hybridizing tothe 3′ end of the primer extension product. Suitable length of theintervening sequence can be empirically determined, and can be at leastabout 1, 2, 4, 6, 8, 10, 12, 15 nucleotides. Suitable sequence identityof the intervening sequence can also be empirically determined, and thesequence is designed to preferably, but not necessarily, enhance degreeof amplification as compared to omission of the sequence. In oneembodiment, the intervening sequence is a sequence that is designed toprovide for enhanced, or more optimal, transcription by the RNApolymerase used. Generally, the sequence is not related (i.e., it doesnot substantially hybridize) to a primer extension product. More optimaltranscription occurs when transcriptional activity of the polymerasefrom a promoter that is operatively linked to said sequence is greaterthan from a promoter that is not so linked. The sequence requirementsfor optimal transcription are generally known in the art as previouslydescribed for various DNA dependent RNA polymerases, such as in U.S.Pat. Nos. 5766849 and 5654142, and can also be empirically determined.

[0208] In another embodiment, the PTO comprises a sequence that is 5′ tothe propromoter sequence, i.e., the PTO comprises additional nucleotides(which may or may not be transcriptional regulatory sequences) located5′ to the propromoter sequence. Generally, but not necessarily, thesequence is not hybridizable (under a given set of conditions) to theprimer extension product.

[0209] In one embodiment, the PTO cannot function efficiently as aprimer for nucleic acid extension. Techniques for blocking the primerfunction of the PTO include any that prevent addition of nucleotides tothe 3′ end of the PTO by a DNA polymerase. Such techniques are known inthe art, including, for example, substitution or modification of the 3′hydroxyl group, or incorporation of a modified nucleotide, such as adideoxynucleotide, in the 3′-most position of the PTO that is notcapable of anchoring addition of nucleotides by a DNA polymerase. It ispossible to block the 3′ end using a label, or a small molecule which isa member of a specific binding pair, such as biotin. It is also possibleto render the 3′ end non-extendable by addition of nucleotides whichcannot hybridize to a primer extension product, either due tonon-complementarity or due to structural modifications which do notsupport hydrogen bonding. In other embodiments, the PTO is not blocked.

[0210] The length of the portion of the PTO that hybridizes to a primerextension product of interest is preferably from about 5 to about 50nucleotides, more preferably from about 10 to about 40 nucleotides, evenmore preferably from about 15 to about 35 nucleotides, and mostpreferably from about 20 to 30 nucleotides. In some embodiments, thehybridizing portion is at least about any of the following: 3, 5, 10,15, 20; and less than about any of the following: 30, 40, 50, 60. Thecomplementarity of the hybridizing portion is preferably at least about25%, more preferably at least about 50%, even more preferably at leastabout 75%, and most preferably at least about 90%, to its intendedbinding sequence on the primer extension product of interest.

DNA Polymerase, Ribonuclease and RNA Polymerase

[0211] The amplification methods of the invention employ the followingenzymes: an RNA-dependent DNA polymerase, a DNA-dependent DNApolymerase, a ribonuclease such as RNase H, and a DNA-dependent RNApolymerase. One or more of these activities may be found and used in asingle enzyme. For example, RNase H activity may be supplied by anRNA-dependent DNA polymerase (such as reverse transcriptase) or may beprovided in a separate enzyme. Reverse transcriptases useful for thismethod may or may not have RNase H activity.

[0212] One aspect of the invention is the formation of double strandedcDNA from a primer-RNA complex. This process generally utilizes theenzymatic activities of an RNA-dependent DNA polymerase, a DNA-dependentDNA polymerase and a ribonuclease activity.

[0213] RNA-dependent DNA polymerases for use in the methods andcompositions of the invention are capable of effecting extension of aprimer according to the methods of the invention. Accordingly, apreferred RNA-dependent RNA polymerase is one that is capable ofextending a nucleic acid primer along a nucleic acid template that iscomprised at least predominantly of ribonucleotides. SuitableRNA-dependent DNA polymerases for use in the methods and compositions ofthe invention include reverse transcriptase. Many reversetranscriptases, such as those from avian myeoloblastosis virus (AMV-RT),and Moloney murine leukemia virus (MMLV-RT) comprise more than oneactivity (for example, polymerase activity and ribonuclease activity)and can function in the formation of the double stranded cDNA molecules.However, in some instances, it is preferable to employ a reversetranscriptase which lacks the RNase H activity. Reverse transcriptasedevoid of RNase H activity are known in the art, including thosecomprising a mutation of the wild type reverse transcriptase where themutation eliminates the RNase H activity. In these cases, the additionof an RNase H from other sources, such as that isolated from E. coli,can be employed for the formation of the double stranded cDNA.

[0214] DNA-dependent DNA polymerases for use in the methods andcompositions of the invention are capable of effecting extension of aprimer according to the methods of the invention. Accordingly, apreferred polymerase is one that is capable of extending a nucleic acidprimer along a nucleic acid template that is comprised at leastpredominantly of deoxynucleotides. The formation of the double strandedcDNA can be carried out by reverse transcriptase which comprises bothRNA-dependent DNA polymerase and DNA-dependent DNA polymeraseactivities. Preferably, the DNA polymerase has high affinity for bindingat the 3′-end of an oligonucleotide hybridized to a nucleic acid strand.Preferably, the DNA polymerase does not possess substantial nickingactivity. Preferably, the polymerase has little or no 5′->3′ exonucleaseactivity so as to minimize degradation of primer, or primer extensionpolynucleotides. Generally, this exonuclease activity is dependent onfactors such as pH, salt concentration, whether the template is doublestranded or single stranded, and so forth, all of which are familiar toone skilled in the art. Mutant DNA polymerases in which the 5′->3′exonuclease activity has been deleted, are known in the art and aresuitable for the amplification methods described herein. Preferably, theDNA polymerase has little to no proofreading activity.

[0215] Suitable DNA polymerases for use in the methods and compositionsof the invention include those disclosed in U.S. Pat. Nos. 5,648,211 and5,744,312, which include exo⁻Vent (New England Biolabs), exo⁻Deep Vent(New England Biolabs), Bst (BioRad), exo⁻Pfu (Stratagene), Bca(Panvera), sequencing grade Taq (Promega), and thermostable DNApolymerases from thermoanaerobacter thermohydrosulfuiricus.

[0216] An agent that cleaves RNA in an RNA/DNA hybrid (e.g.ribonuclease) is used in the methods and compositions of the invention.Preferably, the agent, which can be ribonuclease, cleavesribonucleotides regardless of the identity and type of nucleotidesadjacent to the ribonucleotide to be cleaved. It is preferred that theagent (e.g. ribonuclease) cleaves independent of sequence identity.Examples of suitable ribonucleases for the methods and compositions ofthe invention are well known in the art, including ribonuclease H (RNaseH), including Hybridase.

[0217] The DNA-dependent RNA polymerase for use in the methods andcompositions of the invention are known in the art. Either eukaryotic orprokaryotic polymerases may be used. Examples include T 7, T 3 and SP 6RNA polymerases. Generally, the RNA polymerase selected is capable oftranscribing from the promoter sequence provided by the propromoterpolynucleotides as described herein. Generally, the RNA polymerase is aDNA-dependent polymerase, which is preferably capable of transcribingfrom a single stranded DNA template so long as the promoter region isdouble stranded.

[0218] In general, the enzymes used included in the methods andcompositions of the invention should not produce substantial degradationof the nucleic acid components of said methods and compositions.

Reaction Conditions and Detection

[0219] Appropriate reaction media and conditions for carrying out themethods of the invention are those that permit nucleic acidamplification according to the methods of the invention. Such media andconditions are known to persons of skill in the art, and are describedin various publications, such as U.S. Pat. Nos. 5,554,516; 5,716,785;5,130,238; 5,194,370; 6,090,591; 5,409,818; 5,554,517; 5,169,766;5,480,784; 5,399,491; 5,679,512; and PCT Pub. No. WO 99/42618. Forexample, a buffer may be Tris buffer, although other buffers can also beused as long as the buffer components are non-inhibitory to enzymecomponents of the methods of the invention. The pH is preferably fromabout 5 to about 11, more preferably from about 6 to about 10, even morepreferably from about 7 to about 9, and most preferably from about 7.5to about 8.5. The reaction medium can also include bivalent metal ionssuch as Mg²⁺ or Mn²⁺, at a final concentration of free ions that iswithin the range of from about 0.01 to about 15 mM, and most preferablyfrom about 1 to 10 mM. The reaction medium can also include other salts,such as KCl or NaCl, that contribute to the total ionic strength of themedium. For example, the range of a salt such as KCl is preferably fromabout 0 to about 125 mM, more preferably from about 0 to about 100 mM,and most preferably from about 0 to about 75 mM. The reaction medium canfurther include additives that could affect performance of theamplification reactions, but that are not integral to the activity ofthe enzyme components of the methods. Such additives include proteinssuch as BSA, single stranded binding protein (for example, T4 gene 32protein), and non-ionic detergents such as NP40 or Triton. Reagents,such as DTT, that are capable of maintaining enzyme activities can alsobe included. Such reagents are known in the art. Where appropriate, anRNase inhibitor (such as Rnasin) that does not inhibit the activity ofthe RNase employed in the method can also be included. Any aspect of themethods of the invention can occur at the same or varying temperatures.Preferably, the amplification reactions (particularly, primer extensionand transcription; and generally not the step of denaturing the complexof first and second primer extension products) are performedisothermally, which substantially avoids the cumbersome thermocyclingprocess. The amplification reaction is carried out at a temperature thatpermits hybridization of the oligonucleotides (primer, and/or PTO) ofthe invention to the template polynucleotide and that does notsubstantially inhibit the activity of the enzymes employed. Thetemperature can be in the range of preferably about 25° C. to about 85°C., more preferably about 30° C. to about 80° C., and most preferablyabout 37° C. to about 75° C. The temperature for the transcription stepscan be lower than the temperature(s) for the preceding steps. Thetemperature of the transcription steps can be in the range of preferablyabout 25° C. to about 85° C., more preferably about 30° C. to about 75°C., and most preferably about 37° C. to about 70° C.

[0220] Nucleotide and/or nucleotide analogs, such as deoxyribonucleosidetriphosphates, that can be employed for synthesis of the primerextension products in the methods of the invention are provided in theamount of from preferably about 50 to about 2500 μM, more preferablyabout 100 to about 2000 μM, even more preferably about 200 to about 1700μM, and most preferably about 250 to about 1500 μM. Nucleotides and/oranalogs, such as ribonucleoside triphosphates, that can be employed forsynthesis of the RNA transcripts in the methods of the invention areprovided in the amount of from preferably about 0.25 to about 6 mM, morepreferably about 0.5 to about 5 mM, even more preferably about 0.75 toabout 4 mM, and most preferably about 1 to about 3 mM.

[0221] The oligonucleotide components of the amplification reactions ofthe invention are generally in excess of the number of target nucleicacid sequence to be amplified. They can be provided at about or at leastabout any of the following: 10, 10², 10⁴, 10⁶, 10⁸, 10¹⁰, 10¹² times theamount of target nucleic acid. Primers and PTO can each be provided atabout or at least about any of the following concentrations: 50 nM, 100nM, 500 nM, 1000 nM, 2500 nM, 5000 nM.

[0222] In one embodiment, the foregoing components are addedsimultaneously at the initiation of the amplification process. Inanother embodiment, components are added in any order prior to or afterappropriate timepoints during the amplification process, as requiredand/or permitted by the amplification reaction. Such timepoints, some ofwhich are noted below, can be readily identified by a person of skill inthe art. The enzymes used for nucleic acid amplification according tothe methods of the invention can be added to the reaction mixture eitherprior to the target nucleic acid denaturation step, following thedenaturation step, or following hybridization of the primer to thetarget RNA, as determined by their thermal stability and/or otherconsiderations known to the person of skill in the art. The first strandcDNA (first primer extension product) and the second strand cDNA (secondprimer extension product) synthesis reactions can be performedconsecutively, followed by the amplification steps (for example, bindingof propromoter polynucleotide and transcription). In these embodiments,the reaction conditions and components may be varied between thedifferent reactions.

[0223] The amplification reactions can be stopped at various timepoints,and resumed at a later time. Said timepoints can be readily identifiedby a person of skill in the art. One timepoint is at the end of firststrand cDNA synthesis. Another timepoint is at the end of second strandcDNA synthesis. Methods for stopping the reactions are known in the art,including, for example, cooling the reaction mixture to a temperaturethat inhibits enzyme activity or heating the reaction mixture to atemperature that destroys an enzyme. Methods for resuming the reactionsare also known in the art, including, for example, raising thetemperature of the reaction mixture to a temperature that permits enzymeactivity or replenishing a destroyed (depleted) enzyme. In someembodiments, one or more of the components of the reactions isreplenished prior to, at, or following the resumption of the reactions.Alternatively, the reaction can be allowed to proceed (i.e., from startto finish) without interruption.

[0224] The reaction can be allowed to proceed without purification ofintermediate complexes, for example, to remove primer. Products can bepurified at various timepoints, which can be readily identified by aperson of skill in the art. One timepoint is at the end of first strandcDNA synthesis. Another timepoint is at the end of second strand cDNAsynthesis.

[0225] The detection of the amplification product is indicative of thepresence of the target sequence. Quantitative analysis is also feasible.Direct and indirect detection methods (including quantitation) are wellknown in the art. For example, by comparing the amount of productamplified from a test sample containing an unknown amount of apolynucleotide containing a target sequence to the product ofamplification of a reference sample that has a known quantity of apolynucleotide that contains the target sequence, the amount of targetsequence in the test sample can be determined. The amplification methodsof the invention can also be extended to analysis of sequencealterations and sequencing of the target nucleic acid. Further,detection could be effected by, for example, examination of translationproducts from RNA amplification products.

Compositions and Kits of the Invention

[0226] The invention also provides compositions and kits used in themethods described herein. The compositions may be any component(s),reaction mixture and/or intermediate described herein, as well as anycombination thereof.

[0227] For example, the invention provides compositions comprising: (a)a first primer; (b) a second primer (which can be a random primer); and(c) a propromoter polynucleotide (such as a PTO). In some embodiments,the compositions further comprises: (d) a third primer (which can be arandom primer). In some embodiments, the second primer and/or thirdprimer is a random primer. In some embodiments, the propromoterpolynucleotide ((c), above) is capable of hybridizing to the complementof the 5′ portion of the first primer.

[0228] The invention also provides compositions comprising a propromoterpolynucleotide (such as a PTO) capable of hybridizing to a 3′ portion ofa second primer extension that is complement of a 5′ portion of a firstprimer used to create first primer extension product.

[0229] The invention also provides compositions comprising (a) a firstprimer; (b) a second primer (which can be a random primer); and (c) acomposite primer, wherein the composite primer comprises a 5′ RNAportion and a DNA portion. In some embodiments, the composition furthercomprises one or more of the following: DNA-dependent DNA polymerase,RNA-dependent DNA polymerase, and an agent (generally an enzyme) thatcleaves RNA from an RNA/DNA heteroduplex.

[0230] The invention also provides compositions comprising a propromoterpolynucleotide, wherein the propromoter is hybridizable to a sequence inthe second primer extension product comprising the complement of the 5′portion of a first prime, wherein the first primer is extended to formthe first primer extension product.

[0231] In another aspect, the invention provides complexes and/orreaction intermediates produced (present) in any of the methodsdescribed herein. Examples of such complexes are schematically depictedin FIGS. 1,2 and 3. In one example, a complex of the invention is acomplex comprising: (a) a second or third primer extension product; and(b) a propromoter polynucleotide (for example, a PTO).In yet anotherexample, the invention provides compositions comprising a complex of (a)a third primer extension product; and (b) a propromoter polynucleotide(which can be a PTO). In yet another example, the invention providescompositions comprising a complex of (a) a second primer extensionproduct, generated by denaturation of a hybridized first and secondprimer extension product; and (b) a composite primer hybridizable to thesecond primer extension product.

[0232] The invention also provides compositions comprising theamplification products described herein. Accordingly, the inventionprovides a population of anti-sense RNA molecules which are copies of atarget sequence, which are produced by any of the methods describedherein. The invention also provides a population of anti-sensepolynucleotides (generally DNA) molecules, which are produced by any ofthe methods described herein.

[0233] The compositions are generally in a suitable medium, althoughthey can be in lyophilized form. Suitable media include, but are notlimited to, aqueous media (such as pure water or buffers).

[0234] The invention provides kits for carrying out the methods of theinvention. Accordingly, a variety of kits are provided in suitablepackaging. The kits may be used for any one or more of the usesdescribed herein, and, accordingly, may contain instructions for any oneor more of the following uses: amplifying a RNA sequence of interest,sequencing, genotyping (nucleic acid mutation detection), preparation ofan immobilized nucleic acid (which can be a nucleic acid immobilized ona microarray), characterizing nucleic acids using the amplified nucleicacid products generated by the methods of the invention; gene expressionprofiling, subtractive hybridization; preparation of probes forsubtractive hybridization; and preparing libraries (which can be cDNAand/or differential hybridization libraries).

[0235] The kits of the invention comprise one or more containerscomprising any combination of the components described herein, and thefollowing are examples of such kits. For example, the invention provideskits that comprise a first primer that comprises a sequence that whenintroduced into the amplification steps of the methods of the inventionresults in generation of an intermediate polynucleotide to which apropromoter polynucleotide can hybridize. The invention also provideskits that further comprise a second primer and/or a third primer, eitherof both of which can be a random primer. The kits can contain furthercomponents, such as any of (a) a propromoter polynucleotide (such as aPTO); and (b) any of the enzymes described herein, such as an enzymewhich cleaves RNA from an RNA/DNA hybrid (for example, RNaseH), a DNApolymerase (RNA-dependent or DNA-dependent) or an RNA polymerase. Withrespect to compositions containing a random primer, these compositionsmay also contain a plurality of random primers (i.e., a population ofrandom primers having different sequences).

[0236] Kits may also optionally include any of one or more of theenzymes described herein, as well as deoxynucleoside triphosphatesand/or ribonucleoside triphosphates. Kits may also include one or moresuitable buffers (as described herein). Kits useful for nucleic acidsequencing may optionally include labeled or unlabeled nucleotideanalogs that upon incorporation into a primer extension product effecttermination of nucleotide polymerization. One or more reagents in thekit can be provided as a dry powder, usually lyophilized, includingexcipients, which on dissolution will provide for a reagent solutionhaving the appropriate concentrations for performing any of the methodsdescribed herein. Each component can be packaged in separate containersor some components can be combined in one container wherecross-reactivity and shelf life permit.

[0237] The kits of the invention may optionally include a set ofinstructions, generally written instructions, although electronicstorage media (e.g., magnetic diskette or optical disk) containinginstructions are also acceptable, relating to the use of components ofthe methods of the invention for the intended nucleic acidamplification, and/or, as appropriate, for using the amplificationproducts for purposes such as nucleic acid sequencing and detection ofsequence mutation. The instructions included with the kit generallyinclude information as to reagents (whether included or not in the kit)necessary for practicing the methods of the invention, instructions onhow to use the kit, and/or appropriate reaction conditions. For example,the invention provides kits that comprise a first primer that comprisesa sequence the complement of which is hybridizable by a propromoterpolynucleotide, and instructions for using the primer to amplify RNA. Inanother example, kits can further comprise a second primer and/or athird primer, and optionally instructions for using the primers toamplify RNA. In other examples, the kits can contain further components,such as any of (a) a propromoter polynucleotide (such as a PTO); and (b)any of the enzymes described herein, such as an enzyme which cleaves RNAfrom an RNA/DNA hybrid (for example, RNaseH), DNA polymerase(RNA-dependent or DNA-dependent) and RNA polymerase. In another example,a kit comprises (a) a composite primer; and (b) instructions foramplifying RNA according to any of the methods described herein. In someembodiments, said kit further comprises a PTO. In other embodiments,said kit further comprises one or more of the following components: (a)a first primer; (b) a second primer; (c) an agent (generally RNase H)capable of cleaving RNA from RNA/DNA heteroduplexes; DNA-dependent DNApolymerase; and RNA-dependent DNA-polymerase. Any of these kits canfurther comprise instructions for using the components to amplify RNA.

[0238] The component(s) of the kit may be packaged in any convenient,appropriate packaging. The components may be packaged separately, or inone or multiple combinations. Where kits are provided for practicingamplification methods of the invention, the RNA polymerase (if included)is preferably provided separately from the components used in the stepsprior to the transcription steps.

[0239] The relative amounts of the various components in the kits can bevaried widely to provide for concentrations of the reagents thatsubstantially optimize the reactions that need to occur to practice themethods disclosed herein and/or to further optimize the sensitivity ofany assay.

[0240] The invention also provides systems for effecting the methodsdescribed herein. These systems comprise various combinations of thecomponents discussed above. For example, the invention provides systemsfor amplifying a target RNA, comprising: (a) a first primer; (b) asecond primer (which can be a random primer); (c) an RNA-dependent DNApolymerase; (d) a DNA-dependent DNA polymerase; (e) a propromoterpolynucleotide; and (f) an enzyme that cleaves RNA from an RNA/DNAhybrid. The system may further comprise: (g) a third primer (which canbe a random primer). The system may also further comprise a compositeprimer that hybridizes to a second strand cDNA. A system generallyincludes one or more apparatuses for performing the amplificationmethods of the invention. Such apparatuses include, for example, heatingdevices (such as heating blocks or water baths) and apparatuses whicheffect automation of one or more steps of the methods described herein.

[0241] The invention also provides reaction mixtures (or compositionscomprising reaction mixtures) which contain various combinations ofcomponents described herein. An example of a reaction mixture is (a) acomplex of a first primer extension product and a second primerextension product; (b) a polynucleotide comprising a propromotersequence (for example, a PTO); and (c) RNA polymerase. Other reactionmixtures are described herein and are encompassed by the invention.

Methods Using the Amplification Methods and Compositions of theInvention

[0242] The methods and compositions of the invention can be used for avariety of purposes. For purposes of illustration, methods ofsequencing, genotyping (nucleic acid mutation detection), preparation ofan immobilized nucleic acid (which can be a nucleic acid immobilized ona microarray), and characterizing nucleic acids using the amplifiednucleic acid products generated by the methods of the invention, aredescribed. Methods of expression profiling, methods of subtractivehybridization and the preparation of probes for subtractivehybridization, and methods of preparing libraries (which can be cDNAand/or differential hybridization libraries) are also described.

Sequencing of RNA Targets Using the Methods of the Invention

[0243] The amplification methods of the invention are useful, forexample, for sequencing of an RNA sequence of interest. The sequencingprocess is carried out as described for the amplification methodsdescribed herein.

[0244] The amplification methods of the invention are useful, forexample, for sequencing of an RNA sequence of interest. The sequencingprocess is carried out by amplifying a target RNA containing thesequence of interest by any of the methods described herein. Addition ofnucleotides during primer extension is analyzed using methods known inthe art, for example, incorporation of a terminator nucleotide orsequencing by synthesis (e.g. pyrosequencing).

[0245] In embodiments wherein the end product is in the form ofdisplaced DNA primer extension products, in addition to the nucleotides,such as natural deoxyribonucleotide triphosphates (dNTPs), that are usedin the amplification methods, appropriate nucleotide triphosphateanalogs, which may be labeled or unlabeled, that upon incorporation intoa primer extension product effect termination of primer extension, maybe added to the reaction mixture. Preferably, the dNTP analogs are addedafter a sufficient amount of reaction time has elapsed since theinitiation of the amplification reaction such that a desired amount ofsecond primer extension product or fragment extension product has beengenerated. Said amount of the time can be determined empirically by oneskilled in the art.

[0246] In embodiments wherein the end product is in the form of RNAproducts, sequencing can be based on premature (deliberate) terminationof RNA transcription. The inclusion of rNTP analogs, which may belabeled or unlabeled, that upon incorporation into an RNA transcripteffects termination of rNTP polymerization in the reaction mixture, willresult in production of truncated RNA products, which result fromblocking of the RNA polymerase at sites of incorporation of the analogs.

[0247] Suitable analogs (dNTP and rNTP) include those commonly used inother sequencing methods and are well known in the art. Examples of dNTPanalogs include dideoxyribonucleotides. Examples of rNTP analogs (suchas RNA polymerase terminators) include 3′-dNTP. Sasaki et al.,Biochemistry (1998) 95:3455-3460. These analogs may be labeled, forexample, with fluorochromes or radioisotopes. The labels may also belabels which are suitable for mass spectroscopy. The label may also be asmall molecule which is a member of a specific binding pair, and can bedetected following binding of the other member of the specific bindingpair, such as biotin and streptavidin, respectively, with the lastmember of the binding pair conjugated to an enzyme that catalyzes thegeneration of a detectable signal that could be detected by methods suchas colorimetry, fluorometry or chemiluminescence. All of the aboveexamples are well known in the art. These are incorporated into theprimer extension product or RNA transcripts by the polymerase and serveto stop further extension along a template sequence. The resultingtruncated polymerization products are labeled. The accumulated truncatedproducts vary in length, according to the site of incorporation of eachof the analogs, which represent the various sequence locations of acomplementary nucleotide on the template sequence.

[0248] Analysis of the reaction products for elucidation of sequenceinformation can be carried out using any of various methods known in theart. Such methods include gel electrophoresis and detection of thelabeled bands using appropriate scanner, sequencing gel electrophoresisand detection of the radiolabeled band directly by phosphorescence suchas Molecular Dynamics reader, capillary electrophoresis adapted with adetector specific for the labels used in the reaction, and the like. Thelabel can also be a ligand for a binding protein which is used fordetection of the label in combination with an enzyme conjugated to thebinding protein, such as biotin-labeled chain terminator andstreptavidin conjugated to an enzyme. The label is detected by theenzymatic activity of the enzyme, which generates a detectable signal.

[0249] As with other sequencing methods known in the art, the sequencingreactions for the various nucleotide types (A, C, G, T or U) are carriedout either in a single reaction vessel, or in separate reaction vessels(each representing one of the various nucleotide types). The choice ofmethod to be used is dependent on practical considerations readilyapparent to one skilled in the art, such as the nucleotide tri phosphateanalogs and/or label used. Thus, for example, when each of the analogsis differentially labeled, the sequencing reaction can be carried out ina single vessel. The considerations for choice of reagent and reactionconditions for optimal performance of sequencing analysis according tothe methods of the invention are similar to those for other previouslydescribed sequencing methods. The reagent and reaction conditions shouldbe as described above for the nucleic acid amplification methods of theinvention.

Mutation Detection, Including Mutation Detection Based on SingleStranded Conformation Polymorphism Utilizing the Amplification Methodsof the Invention

[0250] The polynucleotide (generally, RNA and DNA) amplificationproducts generated according to the methods of the invention are alsosuitable for analysis for the detection of any alteration in the targetnucleic acid sequence, as compared to a reference nucleic acid sequencewhich is identical to the target nucleic acid sequence other than thesequence alteration. The sequence alterations may be sequencealterations present in the genomic sequence or may be sequencealterations which are not reflected in the genomic DNA sequences, forexample, alterations due to post transcriptional alterations, and/ormRNA processing, including splice variants.

[0251] The RNA and DNA products of the amplification methods aresuitable for single stranded conformation polymorphism (rSSCP) basedmutation detection. The amplification methods of the invention can bedirectly linked to appropriate means for detecting single strandedconformation polymorphism, such as an electrophoretic separation methodfor the identification of specific mobility pattern of the singlestranded RNA or DNA products for the elucidation of the presence ofspecific sequence feature(s), and/or the presence of any difference in atest nucleic acid as compared to a reference nucleic acid.

[0252] Methods based on gel electrophoresis or capillary electrophoresiscan be used for the detection and analysis of the various singlestranded conformational isomers. Alternatively, it is also likely thatcleavage of the single stranded RNA product using nucleases whichrecognize sequence dependent secondary structures may be useful for thedetermination of sequence specific conformation polymorphism. Suchnucleases are known in the art, such as the Cleavase assay (Third Wave).The electrophoretic methods are potentially more suitable for highthroughput mutation, or genotyping, detection methods.

[0253] The determination of sequence specific electrophoretic patternfor a given nucleic acid sequence is useful for, for example, thedetection of specific alleles of a test sequence. Furthermore, it isexpected that an electrophoretic mobility pattern for the variousalleles could be well differentiated, thus allowing the detection of twoalleles in a nucleic acid sample from a single individual, as requiredfor heterozygous genotype, or multiple alleles. Any alteration in thetest nucleic acid sequence, such as base substitution, insertions ordeletion, could be detected using this method. The method is expected tobe useful for detection of specific single base polymorphism, SNP, andthe discovery of new SNPs. Thus, the invention also provides methods fordetecting a polynucleotide comprising a single nucleotide polymorphism,comprising: (a) amplifying a target polynucleotide using any of themethods described herein; and (b) analyzing the amplification productsfor single stranded conformation, wherein a difference in conformationas compared to a reference single stranded polynucleotide indicates asingle nucleotide polymorphism in the target polynucleotide, whereby apolynucleotide comprising a single nucleotide polymorphism is detected.

[0254] Other art recognized methods of analysis for the detection of anyalteration in the target nucleic acid sequence, as compared to areference nucleic acid sequence, are suitable for use with the singlestranded nucleic acid products of the amplification methods of theinvention. Such methods are well-known in the art, and include variousmethods for the detection of specific defined sequences includingmethods based on allele specific primer extension, allele specific probeligation, differential probe hybridization, and limited primerextension. See, for example, Kurn et al, U.S. Pat. No. 6,251,639 B1;U.S. Pat. Nos. 5,888,819; 6,004,744; 5,882,867; 5,854,033; 5,710,028;6,027,889; 6,004,745; 5,763,178; 5,011,769; 5,185,243; 4,876,187;5,882,867; 5,731,146; WO U.S. 88/02746; WO 99/55912; WO 92/15712; WO00/09745; WO 97/32040; WO 00/56925; and 5,660,988. Thus, the inventionalso provides methods for detection of a mutation in an RNA sequence ofinterest comprising a single nucleotide polymorphism, comprising: (a)amplifying a target RNA using any of the methods described herein; and(b) analyzing the amplification products for presence of an alteration(mutation) as compared to a reference single stranded polynucleotide.

Method of Immobilizing Single Stranded Nucleic Acids

[0255] The single stranded polynucleotide (generally RNA and DNA)products of some of the amplification methods of the invention aresuitable for immobilizing to a surface. The single stranded products areparticularly suitable for preparing microarrays comprising the singlestranded amplification products.

[0256] Amplification products can be immobilized and/or attached to asolid or semi-solid support or surface, which may be made, e.g., fromglass, plastic (e.g., polypropylene, nylon), polyacrylamide,nitrocellulose, or other materials.

[0257] Several techniques are well-known in the art for immobilizingnucleic acids to a solid substrate such as a glass slide. One method isto incorporate modified bases or analogs that contain a moiety that iscapable of attachment to a solid substrate, such as an amine group, aderivative of an amine group or another group with a positive charge,into the amplified nucleic acids. The amplified product is thencontacted with a solid substrate, such as a glass slide, which is coatedwith an aldehyde or another reactive group which will form a covalentlink with the reactive group that is on the amplified product and becomecovalently attached to the glass slide. Microarrays comprising theamplified products can be fabricated using a Biodot (BioDot, Inc.Irvine, Calif.) spotting apparatus and aldehyde-coated glass slides (CELAssociates, Houston, Tex.). Amplification products can be spotted ontothe aldehyde-coated slides, and processed according to publishedprocedures (Schena et al., Proc. Natl. Acad. Sci. U.S.A. (1996), 93:10614-10619). Arrays can also be printed by robotics onto glass, nylon(Ramsay, G., Nature Biotechnol. (1998), 16:40-44), polypropylene(Matson, et al., Anal Biochem. (1995), 224(1): 110-6), and siliconeslides (Marshall, A. and Hodgson, J., Nature Biotechnol. (1998),16:27-31). Other approaches to array assembly include finemicropipetting within electric fields (Marshall and Hodgson, supra), andspotting the polynucleotides directly onto positively coated plates.Methods such as those using amino propyl silicon surface chemistry arealso known in the art, as disclosed at http://www.cmt.corning.com andhttp ://cmgm.stanford.edu/pbrown/.

[0258] One method for making microarrays is by making high-densitypolynucleotide arrays. Techniques are known for rapid deposition ofpolynucleotides (Blanchard et al., Biosensors & Bioelectronics,11:687-690). Other methods for making microarrays, e.g., by masking(Maskos and Southern, Nuc. Acids. Res. (1992), 20:1679-1684), may alsobe used. In principle, and as noted above, any type of array, forexample, dot blots on a nylon hybridization membrane, could be used.However, as will be recognized by those skilled in the art, very smallarrays will frequently be preferred because hybridization volumes willbe smaller.

[0259] The amplified polynucleotides may be spotted as a matrix onsubstrates comprising paper, glass, plastic, polypropylene, nylon,polyacrylamide, nitrocellulose, silicon, optical fiber or any othersuitable solid or semi-solid (e.g., thin layer of polyacrylamide gel(Khrapko, et al., DNA Sequence (1991), 1:375-388) surface.

[0260] An array may be assembled as a two-dimensional matrix on a planarsubstrate or may have a three-dimensional configuration comprising pins,rods, fibers, tapes, threads, beads, particles, microtiter wells,capillaries, cylinders and any other arrangement suitable forhybridization and detection of target molecules. In one embodiment thesubstrate to which the amplification products are attached is magneticbeads or particles. In another embodiment, the solid substrate comprisesan optical fiber. In yet another embodiment, the amplification productsare dispersed in fluid phase within a capillary which, in turn, isimmobilized with respect to a solid phase.

Characterization of Nucleic Acids

[0261] The methods of the invention are particularly amenable toquantitative analysis, as sufficient single stranded polynucleotide(generally, DNA and RNA) products are produced which accurately reflectthe representation of the various mRNA in the starting material. Theamplified products can be analyzed using, for example, probehybridization techniques known in the art, such as Northern blotting,and hybridizing to probe arrays. In addition, the single strandedpolynucleotide products may serve as starting material for otherstarting material for other analytical and/or quantification methodsknown in the art, such as real time PCR, quantitative TaqMan,quantitative PCR using molecular beacons, methods described in Kurn,U.S. Pat. No. 6,251,639, etc. Thus, the invention includes those furtheranalytical and/or quantification methods as applied to any of theproducts of the methods herein.

[0262] In another embodiment, the amplification methods of the inventionare utilized to generate multiple copies of single strandedpolynucleotide (DNA or RNA) products that are labeled by theincorporation of labeled nucleotides during DNA or RNA polymerization.For example, amplification according to the methods of the invention canbe carried out with suitable labeled dNTPs or rNTPs. These labelednucleotides can be directly attached to a label, or can comprise amoiety which could be attached to a label. The label may be attachedcovalently or non-covalently to the amplification products. Suitablelabels are known in the art, and include, for example, a ligand which isa member of a specific binding pair which can be detected/quantifiedusing a detectable second member of the binding pair. Thus,amplification of total mRNA according to the methods of the invention inthe presence of, for example, Cy3-dUTP or Cy5-dUTP results in theincorporation of these nucleotides into the amplification products.

[0263] The labeled amplified products are particularly suitable foranalysis (for example, detection and/or quantification) by contactingthem with, for example, microarrays (of any suitable surface, whichincludes glass, chips, plastic), beads, or particles, that comprisesuitable probes such as cDNA and/or oligonucleotide probes. Thus, theinvention provides methods to characterize (for example, detect and/orquantify) an RNA sequence of interest by generating labeledpolynucleotide (generally, DNA or RNA) products using amplificationmethods of the invention, and analyzing the labeled products. Analysisof labeled products can be performed by, for example, hybridization ofthe labeled amplification products to, for example, probes immobilizedat, for example, specific locations on a solid or semi-solid substrate,probes immobilized on defined particles, or probes immobilized on blots(such as a membrane), for example arrays, which have been describedabove. Other methods of analyzing labeled products are known in the art,such as, for example, by contacting them with a solution comprisingprobes, followed by extraction of complexes comprising the labeledamplification products and probes from solution. The identity of theprobes provides characterization of the sequence identity of theamplified products, and thus by extrapolation the identity of the targetRNA present in a sample. Hybridization of the labeled products isdetectable, and the amount of specific labels that are detected isproportional to the amount of the labeled amplification products of aspecific RNA sequence of interest. This measurement is useful for, forexample, measuring the relative amounts of the various RNA species in asample, which are related to the relative levels of gene expression, asdescribed herein. The amount of labeled products (as indicated by, forexample, detectable signal associated with the label) hybridized atdefined locations on an array can be indicative of the detection and/orquantification of the corresponding target RNA species in the sample.

[0264] The labeled amplified products are particularly suitable foranalysis (for example, detection and/or quantification and/ordetermining presence or absence of) by contacting them with, forexample, microarrays that comprise suitable probes such as cDNA and/oroligonucleotide probes. Thus, the invention provides methods tocharacterize (for example, detect and/or quantify and/or determinepresence or absence of) an RNA sequence of interest by generatinglabeled polynucleotide (generally, RNA or DNA) products usingamplification methods of the invention, and analyzing the labeledproducts. Analysis of labeled products can be performed by, for example,hybridization of the labeled amplification products to, for example,probes immobilized at, for example, specific locations on a solid orsemi-solid substrate, probes immobilized on defined particles, or probesimmobilized on blots (such as a membrane), for example arrays, whichhave been described above. Other methods of analyzing labeled productsare known in the art, such as, for example, by contacting them with asolution comprising probes, followed by extraction of complexescomprising the labeled amplification products and probes from solution.The identity of the probes provides characterization of the sequenceidentity of the amplified products, and thus by extrapolation theidentity of the target RNA present in a sample. Hybridization of thelabeled products is detectable, and the amount of specific labels thatare detected is proportional to the amount of the labeled amplificationproducts of a specific RNA sequence of interest. This measurement isuseful for, for example, measuring the relative amounts of the variousRNA species in a sample, which are related to the relative levels ofgene expression. The amount of labeled products (as indicated by, forexample, detectable signal associated with the label) hybridized atdefined locations on an array can be indicative of the detection and/orquantification and/or presence or absence of the corresponding targetRNA species in the sample.

Determination of Gene Expression Profile

[0265] The amplification methods of the invention are particularlysuitable for use in determining the levels of expression of multiplegenes in a sample since the methods described herein are capable ofamplifying multiple target RNAs in the same sample. As described above,amplification products can be detected and quantified by variousmethods, as described herein and/or known in the art. Since RNA is aproduct of gene expression, the levels of the various RNA species, suchas mRNAs, in a sample is indicative of the relative expression levels ofthe various genes (gene expression profile). Thus, determination of theamount of RNA sequences of interest present in a sample, as determinedby quantifying amplification products of the sequences, provides fordetermination of the gene expression profile of the sample source.

[0266] Accordingly, the invention provides methods of determining geneexpression profile in a sample, said method comprising: amplifyingsingle stranded product from at least one RNA sequence of interest inthe sample, using any of the methods described herein; and determiningamount of amplification products of each RNA sequence of interest,wherein each said amount is indicative of amount of each RNA sequence ofinterest in the sample, whereby the expression profile in the sample isdetermined. Generally, labeled products are generated. In oneembodiment, the target RNA is mRNA. It is understood that amount ofamplification product may be determined using quantitative and/orqualitative methods. Determining amount of amplification productincludes determining whether amplification product is present or absent.Thus, an expression profile can includes information about presence orabsence of one or more RNA sequence of interest. “Absent” or “absence”of product, and “lack of detection of product” as used herein includesinsignificant, or de minimus levels.

[0267] The methods of expression profiling are useful in a wide varietyof molecular diagnostic, and especially in the study of gene expressionin essentially any mammalian cell (including a single cell) or cellpopulation. A cell or cell population (e.g. a tissue) may be from, forexample, blood, brain, spleen, bone, heart, vascular, lung, kidney,pituitary, endocrine gland, embryonic cells, tumors, or the like.Expression profiling is also useful for comparing a control (normal)sample to a test sample, including test samples collected at differenttimes, including before, after, and/or during development, a treatment,and the like.

Method of Preparing a Library

[0268] The single stranded polynucleotides (generally DNA and RNA)products of the methods of the invention are useful in preparinglibraries, including cDNA libraries and subtractive hybridizationlibraries. Using the methods of the invention, libraries may be preparedfrom limited amount of starting material, for example, mRNA extractedfrom limited amount of tissue or even single cells. Accordingly, in oneaspect, the methods of the invention provides preparing a library fromthe single stranded DNA or RNA products of the invention. In stillanother aspect, the invention provides methods for making a library,said method comprising: preparing a subtractive hybridization probeusing any of the methods described herein.

[0269] Accordingly, in one aspect, the methods of the invention providespreparing a library from the single stranded DNA or RNA products of theinvention. In some embodiments, the library is a cDNA library.

Methods of Subtractive Hybridization

[0270] The amplification methods of the invention are particularlysuitable for use in subtractive hybridization methods, since the methodsdescribed herein are capable of amplifying multiple target RNAs in thesame sample, and the methods of the invention are suitable for producinglarge amounts of single stranded anti-sense DNA and RNA product suitablefor use as “driver” in subtractive hybridization. For example, twonucleic acid populations, one sense and one antisense, can be allowed tomix together with one population present in molar excess (“driver”).Sequence present in both populations will form hybrids, while sequencespresent in only one population remain single-stranded. Thereafter,various well known techniques are used to separate the unhybridizedmolecules representing differentially expressed sequences. See, e.g.,Hamson et al., U.S. Pat. No. 5,589,339; Van Gelder, U.S. Pat. No.6,291,170;

[0271] Accordingly, the invention provides methods for performingsubtractive hybridization, said methods comprising: (a) preparingmultiple copies (generally, DNA) of the complement of at least one RNAsequences of interest from a first RNA population using any of theamplification methods described herein; and (b) hybridizing the multiplecopies to a second mRNA population, whereby a subpopulation of thesecond mRNA population forms a complex with a nucleotide DNA copy. Theinvention also provides methods for performing subtractivehybridization, said methods comprising: hybridizing multiple copies ofthe complement of at least one RNA sequences of interest from a firstRNA population using any of the amplification methods described hereinto a second mRNA population, whereby a subpopulation of the second mRNApopulation forms a complex with a copy. In some embodiments, “driver”single stranded anti-sense DNA product of the methods of the inventionis combined with tester (sense) mRNA species. In another aspect, theinvention provides methods of differential amplification in which singlestranded driver (antisense) DNA sequences that hybridize with testermRNA sequence are subjected to cleavage by an agent that cleaves RNApresent in a DNA/RNA hybrid, such as RNase H. Cleavage of the mRNAresults in the inability to generate single stranded DNA product fromthe test mRNA strands. Conversely, non-cleaved tester (i.e., tester mRNAthat did not hybridize to driver DNA molecules) may serve as a substratefor subsequent amplification. Amplified differentially expressedproducts have many uses, including as a differential expression probe,to produce differential expression libraries Accordingly, in anotheraspect, the invention provides methods comprising hybridizing multiplepolynucleotide (generally, DNA) copies of the complement of at least oneRNA sequences of interest from a first RNA population using any of theamplification methods described herein to a second mRNA population,whereby a subpopulation of the second mRNA population forms a complexwith a DNA copy; (b) cleaving RNA in the complex of step (a) with anenzyme that cleaves RNA from an RNA/DNA hybrid; and (c) amplifying anunhybridized subpopulation of the second mRNA population, wherebymultiple copies of single stranded polynucleotide (generally, DNA)complementary to the unhybridized subpopulation of the second mRNApopulation are generated.

[0272] The following Examples are provided to illustrate, but not limit,the invention.

EXAMPLES Example 1 Amplification of Total mRNA

[0273] 0.1 μg of total mRNA is combined with primer 1 (provided at aconcentration of from 0.1 to 1 μM), PTO oligonucleotide (provided at aconcentration of from 0.1 to 1 μM), primer 2 (provided at aconcentration of from 0.1 to 1 μM) in a total volume of about 10 μin abuffer containing 40 mM Tris, pH 8.5, 5 mM DTT, 12 MM MgCl₂, 70 mM KCl,108.8 μg/ml BSA; 1 mM of each dNTP, 2 mM each of RATP, rUTP, rCTP, 1.5mM rGTP, and 0.5 mM rITP). The mixture is incubated at 65° C. for 2minutes, and cooled down to 37° C. (or 42° C.). 10 μl of an enzymemixture containing T 7 RNA polymerase (40 to 80 U), MMLV reversetranscriptase (10 to 30 U), and RNase H (1 to 3 U), is added to thereaction mixture, and the mixture is further incubated for 0.5 to 3hours.

[0274] Aliquots of the reaction mixture are analyzed by gelelectrophoresis (5 to 20% PAGE, Novex) for generation of amplificationproducts.

[0275] Primer 1 sequence:

GACGGATGCGGTCTTTTTTTTN

[0276] “N” denotes a degenerate nucleotide (i.e., it can be A, T, C orG).

[0277] Primer 2:

Random hexamer

[0278] PTO:

ggAATTCTAATACgACTCACTATAgggAgAgCGACGGATGCGGTCT-biotin

[0279] wherein bold letters denote the sequence complementary to the3′-end of the second primer extension product.

[0280] Primer 3:

Primer 3 is the same as primer 1.

EXAMPLE 2 Amplification of Specific mRNA

[0281] The experiment of Example 1 is performed, except that primer 2 issubstituted with a primer specific for a sequence of a defined mRNAspecies to be amplified. For example, the amplification of a sequence ofGAPDH mRNA is carried out with a primer that hybridizes to a site on thefirst cDNA strand (first primer extension product) generated from GAPDHmRNA.

[0282] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications may be practiced. Therefore, thedescriptions and examples should not be construed as limiting the scopeof the invention, which is delineated by the appended claims.

What is claimed is:
 1. A method of generating multiple copies of thecomplementary sequence of an RNA sequence of interest, said methodcomprising the steps of: (a) extending a first primer hybridized to atarget RNA with an RNA-dependent DNA polymerase, whereby a complexcomprising a first primer extension product and the target RNA isproduced; (b) cleaving RNA in the complex of step (b) with an enzymethat cleaves RNA from an RNA/DNA hybrid; (c) extending a second primerhybridized to the first primer extension product with a DNA-dependentDNA polymerase, whereby a complex comprising the first primer extensionproduct and a second primer extension product is produced; (d)denaturing the complex of step (c); and (e) hybridizing to the secondprimer extension product a propromoter polynucleotide comprising apropromoter and a region which hybridizes to the second primer extensionproduct under conditions which allow transcription to occur by RNApolymerase, such that RNA transcripts are produced comprising sequencescomplementary to the target RNA; whereby multiple copies of thecomplementary sequence of the RNA sequence of interest are generated. 2.A method of generating multiple copies of the complementary sequence ofan RNA sequence of interest, said method comprising the steps of: (a)extending a first primer hybridized to a target RNA with anRNA-dependent DNA polymerase, whereby a complex comprising a firstprimer extension product and the target RNA is produced; (b) cleavingRNA in the complex of step (a) with an enzyme that cleaves RNA from anRNA/DNA hybrid; (c) extending a second primer hybridized to the firstprimer extension product with a DNA-dependent DNA polymerase, whereby acomplex comprising the first primer extension product and a secondprimer extension product is produced; (d) denaturing the complex of step(c); (e) hybridizing to the second primer extension product apropromoter polynucleotide comprising a propromoter and a region whichhybridizes to the second primer extension product under conditions whichallow transcription to occur by RNA polymerase, such that RNAtranscripts are produced comprising sequences complementary to thetarget RNA; (f) extending a third primer hybridized to said RNAtranscripts with an RNA-dependent DNA polymerase, whereby a complexcomprising a third primer extension product and an RNA transcript isproduced; (g) cleaving RNA in the complex of step (f) with an enzymethat cleaves RNA from an RNA/DNA hybrid; (h) hybridizing a propromoterpolynucleotide comprising a propromoter and a region which hybridizes toa single stranded third primer extension product under conditions whichallow transcription to occur by RNA polymerase, such that RNAtranscripts are produced comprising sequences complementary to thetarget RNA; (i) optionally repeating steps (f) to (h); whereby multiplecopies of the complementary sequence of the RNA sequence of interest areproduced.
 3. The method of claim 1 or 2, wherein the first primercomprises a 5′ portion that is not hybridizable to the target RNA. 4.The method of claim 3, wherein said 5′ portion comprises a sequence thecomplement of which is hybridizable by the propromoter polynucleotide instep (f).
 5. The method of claim 1 or 2, wherein the target RNA is mRNA.6. The method of claim 1 or 2, wherein the first primer comprises apoly-T sequence.
 7. The method of claim 2, wherein the first primercomprises a 5′ portion that is not hybridizable to the target RNA, andwherein said 5′ portion comprises a sequence the complement of which ishybridizable by the propromoter polynucleotide in step (g).
 8. Themethod of claim 2, wherein the second primer and the third primer arethe same.
 9. The method of claim 2, wherein the second primer and thethird primer are different.
 10. The method of claim 2, wherein thesecond primer and the third primer hybridize to different complementarysequences.
 11. The method of claim 1 or 2, wherein at least one type ofrNTP used is a labeled rNTP, whereby labeled products are generated. 12.The method of claim 1 or 2, wherein the first primer is a random primer.13. The method of claim 1 or 2, wherein the second primer comprises DNA.14. The method of claim 1 or 2, wherein the second primer comprises afragment of the target RNA hybridized to the primer extension product,said fragment generated by cleaving RNA in the complex of step (b) withan enzyme that cleaves RNA from an RNA/DNA hybrid.
 15. The method ofclaims 1 or 2, wherein said method comprises generating multiple copiesof two or more different sequences of interest.
 16. The method of claim15, wherein said method comprises at least two different first primers.17. A method of generating multiple copies of the complementary sequenceof an RNA sequence of interest comprising incubating a reaction mixture,said reaction mixture comprising: (a) a single stranded second primerextension product resulting from step (d) of claim 1; (b) a propromoterpolynucleotide comprising a propromoter and a region which ishybridizable to a single stranded second primer extension product; andan RNA polymerase; wherein the incubation is under conditions thatpermit propromoter polynucleotide hybridization and RNA transcription,whereby multiple copies of the complementary sequence of the RNAsequence of interest are generated.
 18. A method of generating multiplecopies of the complementary sequence of an RNA sequence of interestcomprising incubating a reaction mixture, said reaction mixturecomprising: (a) a single stranded second primer extension productresulting from step (d) of claim 1; (b) a third primer comprising asequence hybridizable to an RNA transcript comprising a sequencecomplementary to the target RNA; (c) a propromoter polynucleotidecomprising a propromoter and a region which is hybridizable to a singlestranded second primer extension product; (d) a propromoterpolynucleotide comprising a propromoter and a region which ishybridizable to a single stranded third primer extension product; (e) anenzyme that cleaves RNA from an RNA/DNA hybrid; and (f) an RNApolymerase; wherein the incubation is under conditions that permitprimer extension, RNA cleavage, propromoter polynucleotide hybridizationand RNA transcription, whereby multiple copies of the complementarysequence of the RNA sequence of interest are generated.
 19. A method ofgenerating multiple copies of the complementary sequence of an RNAsequence of interest, said method comprising incubating a reactionmixture, said reaction mixture comprising: (a) an RNA transcript fromstep (e) of claim 1; (b) a third primer comprising a sequencehybridizable to the RNA transcript; (c) a propromoter polynucleotidecomprising a propromoter and a region which is hybridizable to a singlestranded third primer extension product; (d) an enzyme that cleaves RNAfrom an RNA/DNA hybrid; and (e) an RNA polymerase; wherein theincubation is under conditions that permit primer extension, RNAcleavage, propromoter polynucleotide hybridization and RNAtranscription, whereby multiple copies of the complementary sequence ofthe RNA sequence of interest are generated.
 20. A method of generatingmultiple copies of the complementary sequence of an RNA sequence ofinterest, said method comprising incubating a reaction mixture, saidreaction mixture comprising: (a) a target RNA; (b) a first primercomprising a sequence that is hybridizable to the target RNA; (c) asecond primer comprising a sequence hybridizable to an extension productof the first primer; (d) a propromoter polynucleotide comprising apropromoter and a region which is hybridizable to a single strandedsecond primer extension product; (e) an RNA-dependent DNA polymerase;(f) a DNA-dependent DNA polymerase; (g) an RNA polymerase; and (h) anenzyme that cleaves RNA from an RNA/DNA hybrid; wherein the incubationis under conditions that permit primer hybridization, primer extension,RNA cleavage, propromoter polynucleotide hybridization, and RNAtranscription, whereby multiple copies of the complementary sequence ofthe RNA sequence of interest are generated.
 21. A method of generatingmultiple copies of the complementary sequence of an RNA sequence ofinterest, said method comprising incubating a reaction mixture, saidreaction mixture comprising: (a) a target RNA; (b) a first primercomprising a sequence that is hybridizable to the target RNA; (c) asecond primer comprising a sequence hybridizable to an extension productof the first primer; (d) a third primer comprising a sequencehybridizable to an RNA transcript comprising a sequence complementary tothe target RNA; (e) a propromoter polynucleotide comprising apropromoter and a region which is hybridizable to a single strandedsecond primer extension product; (f) a propromoter polynucleotidecomprising a propromoter and a region which is hybridizable to a singlestranded third primer extension product; (g) an RNA-dependent DNApolymerase; (h) a DNA-dependent DNA polymerase; (i) an RNA polymerase;and (j) an enzyme that cleaves RNA from an RNA/DNA hybrid; wherein theincubation is conditions that permit primer hybridization, primerextension, RNA cleavage, propromoter polynucleotide hybridization, andRNA transcription, whereby multiple copies of the complementary sequenceof the RNA sequence of interest are generated.
 22. The method of claim20 or 21, wherein the first primer comprises a 5′ sequence that is nothybridizable to the target RNA.
 23. The method of claim 22, wherein the5′ sequence comprises a sequence the complement of which is hybridizableby a propromoter polynucleotide.
 24. The method of claim 20 or 21,wherein (a) the first primer comprises a poly-T sequence; and (b) thetarget RNA is mRNA.
 25. The method of claim 21, wherein the secondprimer and the third primer are the same.
 26. The method of claim 21,wherein the second primer and the third primer are different.
 27. Themethod of claim 21, wherein the second primer and the third primerhybridize to different complementary sequences.
 28. The method of any ofclaims 1, 2, 18, 19, 20 and 21, wherein the enzyme that cleaves RNA isRNase H.
 29. The method of any of claims 1, 2, 20 and 21, wherein theRNA-dependent DNA polymerase and DNA-dependent DNA polymerase are oneenzyme.
 30. The method of any of claims 1, 2, 20 and 21, wherein theRNA-dependent DNA polymerase and enzyme that cleaves RNA from an RNA/DNAhybrid are the same enzyme.
 31. The method of any of claims 1, 2, 20 and21, wherein the DNA-dependent DNA polymerase and enzyme that cleaves RNAfrom an RNA/DNA hybrid are the same enzyme.
 32. The method of any ofclaims 1, 2, 20 and 21, wherein the DNA-dependent DNA polymerase, theRNA-dependent DNA polymerase and the enzyme that cleaves RNA from anRNA/DNA hybrid are the same enzyme.
 33. The method of claim 1 or 20,wherein the propromoter polynucleotide comprises a region at the 3′ endwhich hybridizes to the second primer extension product, whereby DNApolymerase extension of the second primer extension product produces adouble stranded promoter from which transcription occurs.
 34. The methodof claim 33, wherein the propromoter polynucleotide is a propromotertemplate oligonucleotide.
 35. The method of claim 2 or 21, wherein thepropromoter polynucleotide comprises a region at the 3′ end whichhybridizes to the third primer extension product, whereby DNA polymeraseextension of the second primer extension product produces a doublestranded promoter from which transcription occurs.
 36. The method ofclaim 35, wherein the propromoter polynucleotide is a propromotertemplate oligonucleotide.
 37. The method of claim 20 or 21, wherein atleast one type of rNTP used is a labeled rNTP, whereby labeled productsare generated.
 38. A method of generating multiple copies of thecomplementary sequence of an RNA sequence of interest, said methodcomprising: (a) hybridizing a composite primer to a single strandedsecond primer extension product resulting from step (d) of claim 1,wherein the composite primer comprises an RNA portion and a 3′ DNAportion; (b) extending the composite primer with a DNA-dependent DNApolymerase, whereby a complex comprising a primer extension product andthe second primer extension product is formed; (c) cleaving RNA in thecomplex of step (b) with an enzyme that cleaves RNA from an RNA/DNAhybrid, such that another composite primer hybridizes to the secondprimer extension product and repeats primer extension by stranddisplacement, whereby multiple copies of the complement of the RNAsequence of interest are produced.
 39. The method of claim 38, whereinthe RNA portion of the composite primer is 5′ with respect to the 3′ DNAportion.
 40. The method of claim 39, wherein the 5′ RNA portion isadjacent to the 3′ DNA portion.
 41. The method of claim 38, wherein thefirst primer comprises the composite primer sequence.
 42. The method ofclaim 38, wherein the composite primer comprises a portion hybridizableto the complement of the 5′ portion of the first primer.
 43. The methodof claim 38, wherein the target RNA is mRNA.
 44. The method of claim 38,wherein said method comprises generating multiple copies of two or moredifferent sequences of interest.
 45. The method of claim 38, wherein theenzyme that cleaves RNA from an RNA/DNA hybrid is RNase H.
 46. Themethod of claim 38, wherein the RNA -dependent DNA polymerase andDNA-dependent DNA polymerase are the same enzyme.
 47. The method ofclaim 38, wherein the RNA-dependent DNA polymerase and enzyme thatcleaves RNA from an RNA/DNA hybrid are the same enzyme.
 48. The methodof claim 38, wherein the DNA-dependent DNA polymerase and enzyme thatcleaves RNA from an RNA/DNA hybrid are the same enzyme.
 49. The methodof claim 38, wherein the DNA-dependent DNA polymerase, the RNA-dependentDNA polymerase and the enzyme that cleaves RNA from an RNA/DNA hybridare the same enzyme.
 50. The method of claim 38, wherein at least onetype of dNTP used is a labeled dNTP, whereby labeled products aregenerated.
 51. A method of sequencing an RNA sequence of interest, saidmethod comprising (a) amplifying a target RNA containing the sequence ofinterest by the method of claim 1 or 20 in the presence of a mixture ofrNTPs and rNTP analogs such that transcription is terminated uponincorporation of an rNTP analog; and (b) analyzing the amplificationproducts to determine sequence.
 52. The method of claim 51, wherein thetarget RNA is mRNA.
 53. A method of sequencing an RNA sequence ofinterest, said method comprising (a) amplifying a target RNA containingthe sequence of interest by the method of claim 2 or 21, wherein RNAtranscripts generated from the second primer extension product areamplified in the presence of a mixture of rNTPs and rNTP analogs suchthat transcription is terminated upon incorporation of an rNTP analog;and (b) analyzing the amplification products to determine sequence. 54.The method of claim 53, wherein the target RNA is mRNA.
 55. A method ofdetecting a mutation in a target RNA, comprising (a) amplifying thetarget RNA by any of the methods of claims 1, 2, 20 and 21; and (b)analyzing the amplification products for the presence of a mutation ascompared to a reference nucleotide.
 56. The method of claim 55, whereinthe target RNA is mRNA.
 57. A method of detecting a mutation in a targetRNA by single stranded conformation polymorphism, comprising (a)amplifying the target RNA by any of the methods of claims 1, 2, 20 and21; and (b) analyzing the amplification products for single strandedconformation, wherein a difference in conformation as compared to areference single stranded polynucleotide indicates a mutation in thetarget polynucleotide.
 58. The method of claim 57, wherein the targetRNA is mRNA.
 59. A method of producing a nucleic acid immobilized to asubstrate comprising (a) amplifying a target RNA by any of the methodsof claims 1, 2, 20 and 21; and (b) immobilizing the amplificationproducts on a substrate.
 60. The method of claim 59, wherein the targetRNA is mRNA.
 61. The method of claim 59, wherein the substrate is amicroarray.
 62. A method of characterizing an RNA sequence of interest,comprising (a) amplifying a target RNA by the method of claim 10; and(b) analyzing the labeled RNA products.
 63. The method of claim 62,wherein step (b) comprises contacting the labeled DNA products with atleast one probe.
 64. The method of claim 63, wherein the at least oneprobe is provided as a microarray.
 65. The method of claim 64, whereinthe microarray comprises at least one probe immobilized on a substratefabricated from a material selected from the group consisting of paper,ceramic, glass, plastic, polypropylene, polystyrene, nylon,polyacrylamide, nitrocellulose, silicon, and optical fiber.
 66. Themethod of claim 65, wherein the probe is immobilized on the substrate ina two-dimensional configuration or a three-dimensional configurationcomprising pins, rods, fibers, tapes, threads, beads, particles,microtiter wells, capillaries, and cylinders.
 67. The method of claim62, wherein the target RNA is mRNA.
 68. The method of claim 62, whereinstep (b) of analyzing the labeled RNA products comprises determiningamount of said products, whereby the amount of the RNA sequence ofinterest present in a sample is quantified.
 69. A method ofcharacterizing an RNA sequence of interest, comprising (a) amplifying atarget RNA by the method of claim 37; and (b) analyzing the labeled RNAproducts.
 70. The method of claim 69, wherein step (b) comprisescontacting the labeled DNA products with at least one probe.
 71. Themethod of claim 70, wherein the at least one probe is provided as amicroarray.
 72. The method of claim 71, wherein the microarray comprisesat least one probe immobilized on a substrate fabricated from a materialselected from the group consisting of paper, glass, plastic,polypropylene, nylon, polyacrylamide, nitrocellulose, silicon, andoptical fiber.
 73. The method of claim 72, wherein the probe isimmobilized on the substrate in a two-dimensional configuration or athree-dimensional configuration comprising pins, rods, fibers, tapes,threads, beads, particles, microtiter wells, capillaries, and cylinders.74. The method of claim 69, wherein the target RNA is mRNA.
 75. Themethod of claim 69, wherein step (b) of analyzing the labeled RNAproducts comprises determining amount of said products, whereby theamount of the RNA sequence of interest present in a sample isquantified.
 76. A method of determining gene expression profile in asample, said method comprising: (a) amplifying at least two RNAsequences of interest in the sample using the method of any of claims 1or 2; and (b) determining amount of amplification products of each RNAsequence of interest, wherein each said amount is indicative of amountof each RNA sequence of interest in the sample, whereby the geneexpression profile in the sample is determined.
 77. The method of claim76, wherein each target RNA is mRNA.
 78. A method of preparing alibrary, said method comprising: (a) amplifying at least two RNAsequences of interest using the method of any of claims 1 and 2; and (b)preparing a library from amplified single stranded DNA or RNA product.79. The method of claim 78, wherein the first primer is a random primer80. The method of claim 78, wherein the first primer comprises a poly-Tportion.
 81. A method of performing subtractive hybridization, saidmethod comprising: (a) preparing multiple DNA copies of the complementof one or more RNA sequences of interest from a first RNA populationusing the methods of claim 38; and (b) hybridizing the multiple copiesto a second mRNA population, whereby a subpopulation of the second mRNApopulation forms a complex with the DNA copies.
 82. A method ofperforming subtractive hybridization, said method comprising: (a)preparing multiple DNA copies of the complement of at least two RNAsequences of interest from a first RNA population using the method ofclaim 38; (b) hybridizing the multiple copies to a second mRNApopulation, whereby a subpopulation of the second mRNA population formsa complex with a DNA copy; (c) cleaving RNA in the complex of step (b)with an enzyme that cleaves RNA from an RNA/DNA hybrid; and (d)amplifying the unhybridized subpopulation of the second mRNA population,whereby multiple copies of single stranded DNA complementary to theunhybridized subpopulation of the second mRNA population are generated.83. A kit comprising a first primer that comprises a sequence thecomplement of which is hybridizable by a propromoter polynucleotide, andinstructions for using the primer to amplify RNA using the methods ofany of claims 1, 2, 17, 18, 19, 20, 21, and
 38. 84. The kit of claim 83,further comprising a second primer.